Conjugates of cell binding molecules with cytotoxic agents

ABSTRACT

A conjugate of a potent cytotoxic agent with a cell-binding molecule having a structure represented by Formula (I), wherein T, L, m, n,  , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , and R 13  are as defined herein, can be used for targeted treatment of cancer, autoimmune disease, and infectious disease.

CROSS REFERENCE OF RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/407,367, filed on May 9, 2019, entitled “CONJUGATES OF CELLBINDING MOLECULES WITH CYTOTOXIC AGENTS,” which in turn is acontinuation of U.S. patent application Ser. No. 14/253,881, filed onApr. 16, 2014, now U.S. Pat. No. 10,399,941, which in turn is acontinuation of PCT/IB2012/053554, filed on Jul. 12, 2012. The entirecontent of each of the prior applications is hereby incorporated byreference.

FIELD OF THE INVENTION

This invention relates to conjugates of potent antimitotic agents with acell-surface receptor binding molecules for targeted therapy. Theinvention also relates to use of the compositions comprising bindingmolecule-antimitotic agent conjugates for treating cancer, autoimmunedisease, and infectious disease.

BACKGROUND OF THE INVENTION

The targeted delivery of highly active cytotoxic drugs by antibodies orother cell-surface receptor binding agents to specific sites of diseasein human body, which in turn can dramatically increase therapeuticwindows of the cytotoxic drugs, has proven a particularly promisingapproach for targeted treatment (Van den Mooter, T. et al Expert OpinBiol Ther. 2015, 15, 749-60). In particular, since US FDA approvals ofAdcetris (brentuximab vedotin) in 2011 and Kadcyla (ado-trastuzumabemtansine) in 2013, almost every major pharmaceutical and biotechcompany has adopted the applications of antibody-drug conjugate (ADC)for targeted treatment of cancers (Chari, R. et al, Angew. Chem., Int.Ed. 2014, 53, 3796-3827; Sievers, E. L. et al. Annu Rev Med. 2013, 64,15-29; Mehrling, T. Future Oncol, 2015, 11, 549). So far, the majorityof ADCs in clinical evaluation utilize the highly potenttubulin-interacting agents, maytansinoids or auristatins. A few ADCs inthe clinic have incorporated other potent effector molecules, such asthe topoisomerase 1 inhibitor SN-38 or the DNA interacting agentscalicheamicin and pyrrolobenzodiazepines (Anderl, J. et al, Methods MolBiol. 2013; 1045:51-70; Thomas, A., et al, Lancet Oncol. 2016 June;17(6):e254-e262).

Several short peptidic compounds that found to have biological activityhave been isolated from natural sources. One of them, Tubulysins(structures shown below), which were the first time isolated by Hofleand Reichenbach et al. (GBF Braunschweig) from a culture growth of themyxobacterial strains of Archangium gephyra (F. Sasse et al. J.Antibiot. 2000, 53, 879-885; WO9813375), are members of group ofantimitotic peptides that inhibit tubulin polymerization in dividingcells, and thus inducing apoptosis. With the exceptional potencyexceeding that of vinblastine, taxol and epothilones (Wipf, et al, Org.Lett. 2004, 6, 4057-60; Peltier, et al, J. Am.

Tubulysin R^(i) R^(ii) R^(iii) A CH₂OCOCH₂CH(CH₃)₂ OCOCH₃ OH BCH₂OCOCH₂CH₂CH₃ OCOCH₃ OH C CH₂OCOCH₂CH₃ OCOCH₃ OH D CH₂OCOCH₂CH(CH₃)₂OCOCH₃ H E CH₂OCOCH₂CH₂CH₃ OCOCH₃ H F CH₂OCOCH₂CH₃ OCOCH₃ H GCH₂OCOCH═CH₂ OCOCH₃ OH H CH₂OCOCH₃ OCOCH₃ H I CH₂OCOCH₃ OCOCH₃ OH U HOCOCH₃ H V H OH H Z H OH OH Pretubulysin CH₃ H H (The structures ofexisting tubulysin compounds)Chem. Soc. 2006, 128, 16018-9; Wipf, et al, Org. Lett., 2007, 9,1605-1607; Wang, et al, Chem. Biol. Drug Des. 2007, 70, 75-86; Pando, etal, Org. Lett. 2009, 11, 5567-9), these antimitotic peptides areexciting leads for targeted therapies. Structurally, the tetrapeptidetubulysins are comprising of N-methylpipecolinic acid (Mep) at theN-terminus, isoleucine (Ile) as the second residue, the uniquethiazole-containing tubuvaline (Tuv) as the third residue, and twopossible γ-amino acids at the C-terminus (tubutyrosine (Tut) ortubuphenylalanine (Tup)). Despite several tubulysins have recently beensynthesized, significant general toxicities (>20% animal body weightloss) of the existing tubulysins at doses required for achieving atherapeutic effect compromise their efficacy (US Patent appl.2010/0048490). We have been interested in the art of a conjugate of acell surface binding ligand, particularly using an antibody to conjugatewith tubulysin derivatives for having significantly lower generaltoxicity, yet useful therapeutic efficiency. Although the naturaltubulysins are ideal payloads for ADCs with their extreme potency intens picomolar ranges of IC₅₀ values against many cell lines, we foundthat the natural tubulysin conjugates were hardly metabolized in animallivers, resulting in severe liver toxicity. A simpler analog, such asusing 2-(dimethylamino)-2-methylpropanoic acid to replace1-methylpiperidine-2-carboxylic acid at the far left side of naturaltubulysin structures did not alternate much potency of the compoundsconjugated to an antibody, but reduced significant liver toxicity of theconjugates. Here this patent discloses these tubulysin conjugates with acell surface binding ligand and using these conjugates for treatingcancer and immune disorders.

SUMMARY OF THE INVENTION

In one illustrative embodiment of the invention provides a conjugate offormula (I):

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   Wherein T is a targeting or binding ligand; L is a releasable        linker;        is a linkage bond that L connects to an atom inside the bracket        independently; n is 1-20 and m is 1-10;    -   Inside the bracket is a potent antimitotic agent wherein R¹, R²,        R³, and R⁴ are independently linear or branched C₁-C₈ of alkyl,        alkylalcohol; C₂-C₈ of heteroalkyl, alkylcycloalkyl,        heterocycloalkyl, alkyl ether, alkyl carboxylate, alkyl amine,        alkyl ester, alkyl amide; C₃-C₈ of aryl, Ar-alkyl, heterocyclic,        carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl;        or two R's: R¹R², R³R⁴, R⁵R⁶, or R¹²R¹³ independently together        form a 3˜7 membered carbocyclic, cycloalkyl, heterocyclic,        heterocycloalkyl, aromatic or heteroaromatic ring system; Y is N        or C; In addition, R¹, R², R³, and R⁴ can be independently        absent;    -   Wherein R⁵, R⁶, R⁸ and R¹⁰ are independently selected from H and        linear or branched C₁-C₄ of alkyl or C₂-C₄ of heteroalkyl;    -   Wherein R⁷ is selected from H, R¹⁴, or —R¹⁴C(═O)X¹R¹⁵;        —R¹⁴X¹R¹⁵; X¹ is selected from O, S, S—S, NH, or NR¹⁴;    -   Wherein R⁹ is H, —O—, —OR¹⁴, —OC(═O)R¹⁴—, —OC(═O)NHR¹⁴—,        —OC(═O)NR¹⁴R¹⁵—, —OC(═O) R¹⁴SSR¹⁵—, OP(═O)(OR¹⁴)—, or        OR¹⁴OP(═O)(OR¹⁵);    -   Wherein R¹¹ is H, R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴C(═O)X²R¹⁶, —R¹⁴X²R¹⁶,        —R¹⁴C(═O)X², wherein X² is —O—, —S—, —NH—, —NHS(O₂), —N(R¹⁴)—,        —O—R¹⁴—, —S—R¹⁴—, —S(═O)—R¹⁴—, or —NHR¹⁴—;    -   Wherein R¹² is H, R¹⁴, —O—, —S—, —N—, ═N—, —OH, —SH, —NH₂, ═NH,        ═NNH₂, —NH(R¹⁴), —OR¹⁴, —C(O)O—, —C(O)OR¹⁶—, —COR¹⁶, —COOR¹⁴—,        C(O)NH—, C(O)NH₂, C(O)NHR¹⁴, —SR¹⁴, —S(═O)R¹⁴, —P(═O)(OR¹⁶)₂,        —OP(═O)(OR¹⁶)₂, —CH₂OP(═O)(OR¹⁶)₂, —SO₂R¹⁶;    -   Wherein R¹³ is linear or branched C₁-C₁₀ of alkyl, alkyl acid,        alkyl amide, alkyl amine; or C₂-C₁₀ of heteroalkyl; or C₃-C₁₀ of        Ar; Ar refers to an aromatic or hetero aromatic group, composed        of one or several rings, comprising four to ten carbon atoms,        preferentially four to six carbon atoms. The term of hetero        aromatic group refers to an aromatic group that has one or        several carbon atoms replaced by hetero atoms, preferentially        one, two or three carbon atoms replaced by O, N, Si, Se, P or S,        more preferentially O, S, N. The term aryl or Ar also refers to        an aromatic group, wherein one or several H atoms can be        replaced independently by R¹⁷, F, Cl, Br, I, OR¹⁶, SR¹⁶,        NR¹⁶R¹⁷, N═NR¹⁶, N═R¹⁶, NR¹⁶R¹⁷, NO₂, SOR¹⁶R¹⁷, SO₂R¹⁶, SO₃R¹⁶,        OSO₃R¹⁶, PR¹⁶R¹⁷, POR¹⁶R¹⁷, PO₂R¹⁶R¹⁷, OP(O)(OR¹⁷)₂,        OCH₂OP(O)(OR¹⁷)₂, OC(O)OP(O)(OR¹⁷)₂, PO(OR¹⁶)(OR¹⁷),        OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂, OC(O)R¹⁷ or OC(O)NHR¹⁷;    -   Wherein R¹⁴ and R¹⁵ are independently H; linear or branched        C₁-C₈ of alkyl; C₂-C₈ of alkenyl, alkynyl, heteroalkyl,        heterocyclic, carbocyclic; C₃-C₈ of aryl, cycloalkyl,        alkylcycloalkyl, heterocycloalkyl, heteroaralkyl,        heteroalkylcycloalkyl, alkylcarbonyl;    -   Wherein when R¹⁴ is bivalent, it is a R¹⁴ that is further        connected to an additional functional group of one to four amino        acid units, or (CH₂CH₂O)_(r), r is an integer ranging from 0 to        12, or C₄-C₁₂ of glycosides, or C₁-C₈ of carboxylic acid;    -   Wherein R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;    -   Wherein R¹⁷ is H, linear or branched C₁-C₈ of alkyl; C₂-C₈ of        alkenyl, alkynyl, heteroalkyl, heterocyclic; C₃-C₈ of aryl,        carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl,        heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄-C₁₂ of        glycosides, or pharmaceutical salts.

In another embodiment, the linker L of the potent antimitoticagent-binding molecule conjugates has the formula: —Ww-(Aa)r-Vv-;wherein: —W— is a Stretcher unit; w is 0 or 1; each -Aa-is independentlyan Amino Acid unit; r is independently an integer ranging from 0 to 12;—V— is a Spacer unit; and v is 0, 1 or 2. The Stretcher unit W mayindependently contain a self-immolative spacer, peptidyl units, ahydrazone bond, disulfide or thiolether bonds.

In another embodiment, the cell-surface binding molecule T may be of anykind presently known, or which become known cell binding ligands, suchas peptides and non-peptides. Generally the binding molecule T is anantibody; a single chain antibody; an antibody fragment that binds tothe target cell; a monoclonal antibody; a single chain monoclonalantibody; or a monoclonal antibody fragment that binds the target cell;a chimeric antibody; a chimeric antibody fragment that binds to thetarget cell; a domain antibody; a domain antibody fragment that binds tothe target cell; adnectins that mimic antibodies; DARPins; a lymphokine;a hormone; a vitamin; a growth factor; a colony stimulating factor; or anutrient-transport molecule (a transferrin); a binding peptide, orprotein, or antibody, or small molecule attached on albumin, polymers,dendrimers, liposomes, nanoparticles, vesicles, (viral) capsids.Preferably the binding molecule T is a monoclonal antibody.

In yet another aspect, a compound of formula I˜VII or a pharmaceuticallyacceptable salt or solvate thereof is used for treating cancer, anautoimmune disease or an infectious disease in a human or an animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general synthesis of Tuv component of a Tubulysinanalog.

FIG. 2 shows the synthesis of tubulysin components.

FIG. 3 shows the synthesis of tubulysin components.

FIG. 4 shows the synthesis of components of tubulysin analogs.

FIG. 5 shows the synthesis of components of tubulysin analogs.

FIG. 6 shows the synthesis of components of tubulysin analogs.

FIG. 7 shows the synthesis of components of tubulysin analogs.

FIG. 8 shows the synthesis of components of tubulysin analogs containinga conjugate linker.

FIG. 9 shows the synthesis of components of tubulysin analogs and theirconjugations to an antibody.

FIG. 10 shows the synthesis of components of a linker.

FIG. 11 shows the synthesis of Tubulysin analogs containing a linker andtheir conjugations to an antibody.

FIG. 12 shows the synthesis of Tubulysin analogs containing a linker andtheir conjugations to an antibody.

FIG. 13 shows the synthesis of Tubulysin analogs containing a linker andtheir conjugations to an antibody.

FIG. 14 shows the synthesis of tubulysin analogs containing a linker andtheir conjugations to an antibody.

FIG. 15 shows the synthesis of components of a linker and their linkageto Tubulysin analogs containing a linker as well the conjugation to anantibody.

FIG. 16 shows the synthesis of components of Tup and Tuv analogs.

FIG. 17 shows the synthesis of components of Tuv analogs.

FIG. 18 shows the synthesis of tubulysin analogs containing a linker andtheir conjugations to an antibody.

FIG. 19 shows the synthesis of tubulysin analogs containing a linker andtheir conjugations to an antibody.

FIG. 20 shows the synthesis of components of Tubulysin analogs.

FIG. 21 shows the synthesis of components of Tubulysin analogs.

FIG. 22 shows the comparison of the anti-tumor effect of conjugatecompounds C-166a, C-719, C-720, and C-723 with T-DM1 using human gastrictumor N87 cell model, i.v., one injection at dosing of 6 mg/kg forconjugates C-166a, C-719, C-720, C-723 and T-DM1. Four conjugates testedhere demonstrated better anti-tumor activity than T-DM1. All 6/6 animalsat the groups of compounds C-166a, C-719, C-720, and C-723 hadcompletely no tumor measurable at day 22 till day 36, and all of themcan inhibit the tumor growth for over 48 days. In contrast T-DM1 at doseof 6 mg/Kg was not able to eliminate the tumors and it only inhibitedthe tumor growth for 31 days.

FIGS. 23A and 23B shows an acute toxicity study on ADC conjugates T-DM1,C-166a, C-719, C-720, and C-723 through observing changes in body weight(BW) of mice treated with dose of 75 mg/Kg (FIG. 23A) and 150 mg/Kg(FIG. 23B) in 12 days. The body weight changes demonstrated thatconjugate C-723 was more toxic at both doses than T-DM1; conjugate C-720was similar toxic to T-DM1 at dose of 75 mg/Kg and less toxic than T-DM1at dose of 150 mg/Kg; and both conjugate C-166a and conjugate C-719 aremuch less toxic than T-DM1 at both the tested doses.

FIGS. 24A and 24B show the liver pathogen of the mice treated with doseof 75 mg/Kg of conjugate compounds T-DM1, C-166a, C-719, C-720, andC-723 in comparison with PBS buffer on day 5. The pictures were enlargedby 40 fold. As the pictures indicated: (1). T-DM1 group (in FIG. 24A),pathology of T-DM1 75 mg/kg group indicated hepatocyte swelling andmultifocal necrosis. The lobule structures were not clear. The centralvenules contained the swollen hepatocytes, red blood cells andred-colored remaining. The nucleuses of hepatocyte were in differentsizes and stains. Hepatocytes exhibited the blurred boundaries,increased volume, and eosinophilic-stained plasma. Part of the livernucleus disappeared. An obvious proliferative phase was seen; (2) InC-723 group (in FIG. 24B), scattered single cell necrosis andwater-degeneration are main pathological behaviors. In swelling area,hepatic lobule structure is lost, and a large number of red blood cellsare congested in the central venules. Hepatocytes are swollen,borderline unclear and eosinophilic staining. The nuclei vary in sizesand colors. Mild proliferation is observed. (3). Pathology in C-720group (in FIG. 24C) exhibits the exudate in the central vein of thelobule, disorder in plates arrangement of hepatocytes and hepatocytehyperplasia. Hypertrophies of Kupffer's cells were occasionallyobserved. (4). in both C-719 ((in FIG. 24D) and C-166a (in FIG. 24E)groups, hepatic lobular structure was slightly disordered. Hepaticsinuses were visible. Inflammatory cell infiltration was observed in thewall of the bile ducts. Hypertrophy of Kupffer's cells was rare.Hepatocytes were mildly swollen. The microscopic structure is similar towhat was seen in the control PBS group (in FIG. 24F).

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Alkyl” refers to an aliphatic hydrocarbon group or univalent groupsderived from alkane by removal of one or two hydrogen atoms from carbonatoms. It may be straight or branched having C₁-C₈ (1 to 8 carbon atoms)in the chain. “Branched” means that one or more lower C numbers of alkylgroups such as methyl, ethyl or propyl are attached to a linear alkylchain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl,cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl,3-methyl-hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl,3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl,n-heptyl, isoheptyl, n-octyl, and isooctyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from —C₁-C₈ alkyl and aryl.

“Halogen” refers to fluorine, chlorine, bromine or iodine atom;preferably fluorine and chlorine atom.

“Heteroalkyl” refers to C₂-C₈ alkyl in which one to four carbon atomsare independently replaced with a heteroatom from the group consistingof O, S and N.

“Carbocycle” refers to a saturated or unsaturated ring having 3 to 8carbon atoms as a monocycle or 7 to 13 carbon atoms as a bicycle.Monocyclic carbocycles have 3 to 6 ring atoms, more typically 5 or 6ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, arranged as abicycle [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicycle [5,6] or [6,6] system. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl.

A “C₃-C₈ carbocycle” refers to a 3-, 4-, 5-, 6-, 7- or 8-memberedsaturated or unsaturated nonaromatic carbocyclic ring. A C₃-C₈carbocycle group can be unsubstituted or substituted with one or moregroups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),-aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —SR′, —S(O)R′, —S(O)₂R′, —OH, -halogen, —N₃, —NH₂, —NH(R′),—N(R′)₂ and —CN; where each R′ is independently selected from —C₁-C₈alkyl and aryl.

“Alkenyl” refers to an aliphatic hydrocarbon group containing acarbon-carbon double bond which may be straight or branched having 2 to8 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl,propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl,hexylenyl, heptenyl, octenyl.

“Alkynyl” refers to an aliphatic hydrocarbon group containing acarbon-carbon triple bond which may be straight or branched having 2 to8 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl,propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentynyl,hexylynyl, heptynyl, and octynyl.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—), 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene,propargyl and 4-pentynyl.

“Aryl” or Ar refers to an aromatic or hetero aromatic group, composed ofone or several rings, comprising three to fourteen carbon atoms,preferentially six to ten carbon atoms. The term of “hetero aromaticgroup” refers one or several carbon on aromatic group, preferentiallyone, two, three or four carbon atoms are replaced by O, N, Si, Se, P orS, preferentially by O, S, and N. The term aryl or Ar also refers to anaromatic group, wherein one or several H atoms are replacedindependently by —R′, -halogen, —OR′, or —SR′, —NR′R″, —N═NR′, —N═R′,—NR′R″, —NO₂, —S(O)R′, —S(O)₂R′, —S(O)₂OR′, —OS(O)₂OR′, —PR′R″,—P(O)R′R″, —P(OR′)(OR″), —P(O)(OR′)(OR″) or —OP(O)(OR′)(OR″) wherein R′,R″ are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl,arylalkyl, carbonyl, or pharmaceutical salts.

“Heterocycle” refers to a ring system in which one to four of the ringcarbon atoms are independently replaced with a heteroatom from the groupof O, N, S, Se, B, Si and P. Preferable heteroatoms are O, N and S.Heterocycles are also described in The Handbook of Chemistry andPhysics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226, thedisclosure of which is hereby incorporated by reference. Preferrednonaromatic heterocyclic include epoxy, aziridinyl, thiiranyl,pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl,tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl,piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl,pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl,thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl,tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl,dihydrothiopyranyl, azepanyl, as well as the fused systems resultingfrom the condensation with a phenyl group.

The term “heteroaryl” or aromatic heterocycles refers to a 3 to 14,preferably 5 to 10 membered aromatic hetero, mono-, bi-, or multi-cyclicring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl,pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl,imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl,1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl,benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl,isoxazolyl, pyridyl-N-oxide, as well as the fused systems resulting fromthe condensation with a phenyl group.

“Alkyl”, “cycloalkyl”, “alkenyl”, “alkynyl”, “aryl”, “heteroaryl”,“heterocyclic” and the like refer also to the corresponding “alkylene”,“cycloalkylene”, “alkenylene”, “alkynylene”, “arylene”, “heteroarylene”,“heterocyclene” and the likes which are formed by the removal of twohydrogen atoms.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Examples ofheteroarylalkyl groups are 2-benzimidazolylmethyl, 2-furylethyl.

Examples of a “hydroxyl protecting group” includes, methoxymethyl ether,2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether,p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether,triisopropylsilyl ether, t-butyldimethylsilyl ether,triphenylmethylsilyl ether, acetate ester, substituted acetate esters,pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Such leaving groups are well known in the art,and examples include, a halide (e.g., chloride, bromide, and iodide),methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate. Apreferred leaving group is selected from nitrophenol;N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol;tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol;triflate; imidazole; dichlorophenol; tetrachlorophenol;1-hydroxybenzotriazole; tosylate; mesylate;2-ethyl-5-phenylisoxazolium-3′-sulfonate, anhydrides formed its self, orformed with the other anhydride, e.g. acetyl anhydride, formylanhydride; or an intermediate molecule generated with a condensationreagent for peptide coupling reactions or for Mitsunobu reactions.

The following abbreviations may be used herein and have the indicateddefinitions: Boc, tert-butoxy carbonyl; BroP,bromotrispyrrolidinophosphonium hexafluorophosphate; CDI,1,1′-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE,dichloroethane; DCM, dichloromethane; DEAD is diethyl azodicarboxylate,DIAD, diisopropylazodicarboxylate; DIBAL-H, diisobutyl-aluminiumhydride; DIPEA or DEA, diisopropylethylamine; DEPC, diethylphosphorocyanidate; DMA, N,N-dimethyl acetamide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO,dimethylsulfoxide; DTPA is diethylenetriaminepentaacetic acid; DTT,dithiothreitol; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride; ESI-MS, electrospray mass spectrometry; EtOAc is ethylacetate; Fmoc is N-(9-fluorenylmethoxycarbonyl); HATU,O-(7-azabenzotriazol-1-yl)-N, N, N′, N′-tetramethyluroniumhexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high pressureliquid chromatography; NHS, N-Hydroxysuccinimide; MeCN is acetonitrile;MeOH is methanol; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS,phosphate-buffered saline (pH 7.0-7.5); Ph is phenyl; phe isL-phenylalanine; PyBrop is bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate; PEG, polyethylene glycol; SEC, size-exclusionchromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA,trifluoroacetic acid; THF, tetrahydrofuran; Val, valine; TLC is thinlayer chromatography; UV is ultraviolet.

The “amino acid(s)” can be natural and/or unnatural amino acids,preferably alpha-amino acids. Natural amino acids are those encoded bythe genetic code, which are alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tyrosine. tryptophan and valine. The unnatural amino acidsare derived forms of proteinogenic amino acids. Examples includehydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine,gamma-aminobutyric acid (the neurotransmitter), ornithine, citrulline,beta alanine (3-aminopropanoic acid), gamma-carboxyglutamate,selenocysteine (present in many noneukaryotes as well as mosteukaryotes, but not coded directly by DNA), pyrrolysine (found only insome archaea and one bacterium), N-formylmethionine (which is often theinitial amino acid of proteins in bacteria, mitochondria, andchloroplasts), 5-hydroxytryptophan, L-dihydroxyphenylalanine,triiodothyronine, L-3,4-dihydroxyphenylalanine (DOPA), andO-phosphoserine. The term amino acid also includes amino acid analogsand mimetics. Analogs are compounds having the same general H₂N(R)CHCO₂Hstructure of a natural amino acid, except that the R group is not onefound among the natural amino acids. Examples of analogs includehomoserine, norleucine, 3-aminopropanoic acid, 4-aminobutanoic acid,5-aminopentanoic acid, 6-aminohexanoic acid, 7-aminoheptanoic acid,methionine-sulfoxide, and methionine methyl sulfonium. Preferably, anamino acid mimetic is a compound that has a structure different from thegeneral chemical structure of an alpha-amino acid but functions in amanner similar to one. The term “unnatural amino acid” is intended torepresent the “D” stereochemical form, the natural amino acids being ofthe “L” form. When 1-8 amino acids are used in this patent application,amino acid sequence is then preferably a cleavage recognition sequencefor a protease. Many cleavage recognition sequences are known in theart. See, e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al.Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175(1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth.Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994);and Bouvier et al. Meth. Enzymol. 248: 614 (1995); the disclosures ofwhich are incorporated herein by reference. In particular, the sequenceis selected from the group consisting of Val-Cit, Ala-Val, Ala-Ala,Val-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit,Val-Lys, Ala-Ala-Asn, Asp-Lys, Asp-Glu, Glu-Lys, Lys, Cit, Ser, and Glu.

The “glycoside” is a molecule in which a sugar group is bonded throughits anomeric carbon to another group via a glycosidic bond. Glycosidescan be linked by an O- (an O-glycoside), N- (a glycosylamine), S- (athioglycoside), or C- (a C-glycoside) glycosidic bond. Its core theempirical formula is C_(m)(H₂O)_(n) (where m could be different from n,and m and n are <36), Glycoside herein includes glucose (dextrose),fructose (levulose) allose, altrose, mannose, gulose, iodose, galactose,talose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine,sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), ribose, arabinose,xylose, lyxose, sorbitol, mannitol, sucrose, lactose, maltose,trehalose, maltodextrins, raffinose, Glucuronic acid (glucuronide), andstachyose. It can be in D form or L form, 5 atoms cyclic furanose forms,6 atoms cyclic pyranose forms, or acyclic form, α-isomer (the —OH of theanomeric carbon below the plane of the carbon atoms of Haworthprojection), or a β-isomer (the —OH of the anomeric carbon above theplane of Haworth projection). It is used herein as a monosaccharide,disaccharide, polyols, or oligosaccharides containing 3-6 sugar units.

The term “antibody,” as used herein, refers to a full-lengthimmunoglobulin molecule or an immunologically active portion of afull-length immunoglobulin molecule, i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. Preferably, however, the immunoglobulin is of human, murine, orrabbit origin. Antibodies useful in the invention are preferablymonoclonal, and include, but are not limited to, polyclonal, monoclonal,bispecific, human, humanized or chimeric antibodies, single chainantibodies, Fv, Fab fragments, F(ab′) fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, CDR's, and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens.

An “enantiomer”, also known as an “optical isomer”, is one of twostereoisomers that are mirror images of each other that arenon-superposable (not identical), much as one's left and right hands arethe same except for being reversed along one axis (the hands cannot bemade to appear identical simply by reorientation). A single chiral atomor similar structural feature in a compound causes that compound to havetwo possible structures which are non-superposable, each a mirror imageof the other. The presence of multiple chiral features in a givencompound increases the number of geometric forms possible, though theremay be some perfect-mirror-image pairs. Enantiopure compounds refer tosamples having, within the limits of detection, molecules of only onechirality. When present in a symmetric environment, enantiomers haveidentical chemical and physical properties except for their ability torotate plane-polarized light (+/−) by equal amounts but in oppositedirections (although the polarized light can be considered an asymmetricmedium). They are sometimes called optical isomers for this reason. Amixture of equal parts of an optically active isomer and its enantiomeris termed racemic and has zero net rotation of plane-polarized lightbecause the positive rotation of each (+) form is exactly counteractedby the negative rotation of a (−) one. Enantiomer members often havedifferent chemical reactions with other enantiomer substances. Sincemany biological molecules are enantiomers, there is sometimes a markeddifference in the effects of two enantiomers on biological organisms. Indrugs, for example, often only one of a drug's enantiomers isresponsible for the desired physiologic effects, while the otherenantiomer is less active, inactive, or sometimes even productive ofadverse effects. Owing to this discovery, drugs composed of only oneenantiomer (“enantiopure”) can be developed to enhance thepharmacological efficacy and sometimes eliminate some side effects.

Isotopes are variants of a particular chemical element which differs inneutron number. All isotopes of a given element have the same number ofprotons in each atom. Each atomic number identifies a specific element,but not the isotope; an atom of a given element may have a wide range inits number of neutrons. The number of nucleons (both protons andneutrons) in the nucleus is the atom's mass number, and each isotope ofa given element has a different mass number. For example, carbon-12,carbon-13 and carbon-14 are three isotopes of the element carbon withmass numbers 12, 13 and 14 respectively. The atomic number of carbon is6, which means that every carbon atom has 6 protons, so that the neutronnumbers of these isotopes are 6, 7 and 8 respectively. Hydrogen atom hasthree isotopes of protium (¹H), deuterium (²H), and tritium (³H), whichdeuterium has twice the mass of protium and tritium has three times themass of protium. Isotopic substitution can be used to determine themechanism of a chemical reaction and via the kinetic isotope effect.Isotopic substitution can be used to study how the body affects aspecific xenobiotic/chemical after administration through the mechanismsof absorption and distribution, as well as the metabolic changes of thesubstance in the body (e.g. by metabolic enzymes such as cytochrome P450or glucuronosyltransferase enzymes), and the effects and routes ofexcretion of the metabolites of the drug. This study is calledpharmacokinetics (PK). Isotopic substitution can be used to study of thebiochemical and physiologic effects of drugs. The effects can includethose manifested within animals (including humans), microorganisms, orcombinations of organisms (for example, infection). This study is calledpharmacodynamics (PD). The effects can include those manifested withinanimals (including humans), microorganisms, or combinations of organisms(for example, infection). Both together influence dosing, benefit, andadverse effects of the drug. isotopes can contain a stable(non-radioactive) or an unstable element. Isotopic substitution of adrug may have a different therapeutical efficacy of the original drug.

“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate.

“Pharmaceutically acceptable solvate” or “solvate” refer to anassociation of one or more solvent molecules and a disclosed compound.Examples of solvents that form pharmaceutically acceptable solvatesinclude, but are not limited to, water, isopropanol, ethanol, methanol,DMSO, ethyl acetate, acetic acid and ethanolamine.

“Pharmaceutically acceptable excipient” includes any carriers, diluents,adjuvants, or vehicles, such as preserving or antioxidant agents,fillers, disintegrating agents, wetting agents, emulsifying agents,suspending agents, solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions as suitabletherapeutic combinations.

As used herein, “pharmaceutical salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. The pharmaceutically acceptable saltsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. For example, such conventional non-toxicsalts include those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; andthe salts prepared from organic acids such as acetic, propionic,succinic, tartaric, citric, methanesulfonic, benzenesulfonic,glucuronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic,fumaric, maleic, lactic and the like. Further addition salts includeammonium salts such as tromethamine, meglumine, epolamine, etc., metalsalts such as sodium, potassium, calcium, zinc or magnesium.

The pharmaceutical salts of the present invention can be synthesizedfrom the parent compound which contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared viareaction the free acidic or basic forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two. Generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,PA, 1985, p. 1418, the disclosure of which is hereby incorporated byreference.

“Administering” or “administration” refers to any mode of transferring,delivering, introducing or transporting a pharmaceutical drug or otheragent to a subject. Such modes include oral administration, topicalcontact, intravenous, intraperitoneal, intramuscular, intralesional,intranasal, subcutaneous or intrathecal administration. Alsocontemplated by the present invention is utilization of a device orinstrument in administering an agent. Such device may utilize active orpassive transport and may be slow-release or fast-release deliverydevice.

In the context of cancer, the term “treating” includes any or all of:preventing growth of tumor cells or cancer cells, preventing replicationof tumor cells or cancer cells, lessening of overall tumor burden andameliorating one or more symptoms associated with the disease.

In the context of an autoimmune disease, the term “treating” includesany or all of: preventing replication of cells associated with anautoimmune disease state including, but not limited to, cells capable ofproducing an autoimmune antibody, lessening the autoimmune-antibodyburden and ameliorating one or more symptoms of an autoimmune disease.

In the context of an infectious disease, the term “treating” includesany or all of: preventing the growth, multiplication or replication ofthe pathogen that causes the infectious disease and ameliorating one ormore symptoms of an infectious disease.

Examples of a “mammal” or “animal” include, but are not limited to, ahuman, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat,bird and fowl.

Drug-Linker—Binding Ligand Conjugates

As stated above, this invention provides a cell surface bindingmolecule—antimitotic (cytotoxic) agent conjugate of Formula (I):

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   Wherein T is a targeting or binding ligand; L is a releasable        linker;        is a linkage bond that L connects to an atom inside the bracket        independently; n is 1˜20 and m is 1˜10; Inside the bracket is a        potent antimitotic agent wherein R¹, R², R³, and R⁴ are        independently linear or branched C₁-C₈ of alkyl, alkylalcohol;        C₂-C₈ of heteroalkyl, alkylcycloalkyl, heterocycloalkyl, alkyl        ether, alkyl carboxylate, alkyl amine, alkyl ester, alkyl amide;        C₃-C₈ of aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl,        heteroalkylcycloalkyl, alkylcarbonyl; or two R's: R¹R², R³R⁴, R⁵        R⁶, or R¹²R¹³ together independently form a 3-7 membered        carbocyclic, cycloalkyl, heterocyclic, heterocycloalkyl,        aromatic or heteroaromatic ring system; Y is N or C; In        addition, R¹, R², R³, and R⁴ can be independently absent;    -   Wherein R⁵, R⁶, R⁸ and R¹⁰ are independently selected from H and        linear or branched C₁-C₄ of alkyl or C₂-C₄ of heteroalkyl;    -   Wherein R⁷ is selected from H, R¹⁴, or —R¹⁴C(═O)X¹R¹⁵;        —R¹⁴X¹R¹⁵; X¹ is selected from O, S, S—S, NH, or NR¹⁴;    -   Wherein R⁹ is H, —O—, —OR¹⁴, —OC(═O)R¹⁴—, —OC(═O)NHR¹⁴—,        —OC(═O)NR¹⁴R¹⁵—, —OC(═O) R¹⁴SSR¹⁵—, OP(═O)(OR¹⁴)—, or        OR¹⁴OP(═O)(OR¹⁵);    -   Wherein R¹¹ is H, R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴C(═O)X²R¹⁶, —R¹⁴X²R¹⁶,        —R¹⁴C(═O)X², wherein X² is —O—, —S—, —NH—, —NHS(O₂), —NHS(O),        —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—, —S(═O)—R¹⁴—, or —NHR¹⁴—;    -   Wherein R¹² is H, R¹⁴, —O—, —S—, —N—, ═N—, ═NNH—, —OH, —SH,        —NH₂, ═NH, ═NNH₂, —NH(R¹⁴), —OR¹⁴, —C(O)O—, —C(O)OR¹⁶—, —COR¹⁶,        —COOR¹⁴—, C(O)NH—, C(O)NH₂, C(O)NHR¹⁴, —SR¹⁴, —S(═O)R¹⁴,        —P(═O)(OR¹⁶)₂, —OP(═O)(OR¹⁶)₂, —CH₂OP(═O)(OR¹⁶)₂, —SO₂R¹⁶;    -   Wherein R¹³ is linear or branched C₁-C₁₀ of alkyl, alkyl acid,        alkyl amide, alkyl amine; or C₂-C₁₀ of heteroalkyl; or C₃-C₁₀ of        Ar; Ar refers to an aromatic or hetero aromatic group, composed        of one or several rings, comprising four to ten carbon atoms,        preferentially four to six carbon atoms. The term of hetero        aromatic group refers to an aromatic group that has one or        several carbon atoms replaced by hetero atoms, preferentially        one, two or three carbon atoms replaced by O, N, Si, Se, P or S,        more preferentially O, S, N. The term aryl or Ar also refers to        an aromatic group, wherein one or several H atoms can be        replaced independently by R¹⁷, F, Cl, Br, I, OR¹⁶, SR¹⁶,        NR¹⁶R¹⁷, N═NR¹⁶, N═R¹⁶, NR¹⁶R¹⁷, NO₂, SOR¹⁶R¹⁷, SO₂R¹⁶, SO₃R¹⁶,        OSO₃R¹⁶, PR¹⁶R¹⁷, POR¹⁶R¹⁷, PO₂R¹⁶R¹⁷, OP(O)(OR¹⁷)₂,        OCH₂OP(O)(OR¹⁷)₂, OC(O)OP(O)(OR¹⁷)₂, PO(OR¹⁶)(OR¹⁷),        OP(O)(OR⁷)OP(O)(OR¹⁷)₂, OC(O)R¹⁷ or OC(O)NHR¹⁷;    -   Wherein R¹⁴ and R¹⁵ are independently H; linear or branched        C₁-C₈ of alkyl; C₂-C₈ of alkenyl, alkynyl, heteroalkyl,        heterocyclic, carbocyclic; C₃-C₈ of aryl, cycloalkyl,        alkylcycloalkyl, heterocycloalkyl, heteroaralkyl,        heteroalkylcycloalkyl, alkylcarbonyl;    -   Wherein when R¹⁴ is bivalent, it is a R¹⁴ that is further        connected to an additional functional group of one to four amino        acid units, or (CH₂CH₂O)_(r), r is an integer ranging from 0 to        12, or C₄-C₁₂ of glycosides, or C₁-C₈ of carboxylic acid;    -   Wherein R¹⁶ is H, OH, R¹⁴ or one to four amino acid units;    -   Wherein R¹⁷ is H, linear or branched C₁-C₈ of alkyl; C₂-C₈ of        alkenyl, alkynyl, heteroalkyl, heterocyclic; C₃-C₈ of aryl,        carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl,        heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄-C₁₂ of        glycosides, or pharmaceutical salts.

In another embodiment, conjugates of antimitotic agents have the formula(II)

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   Wherein T, L, n, m, Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R¹⁰, R¹³,        R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are defined the same as in the Formula        (I);    -   Wherein R⁷ is selected from H, R¹⁴, or —R¹⁴C(═O)X¹R¹⁵;        —R¹⁴X¹R¹⁵; X¹ is selected from O, S, S—S, NH, or NR¹⁴;    -   Wherein R⁹ is H, —OH, —OR¹⁴, —OC(═O)R¹⁴, —OC(═O)NHR¹⁴,        —OC(═O)NR¹⁴R¹⁵, —OC(═O) R¹⁴SSR¹⁵, OP(═O)(OR¹⁴)₂, or        OR¹⁴OP(═O)(OR¹⁵);    -   Wherein R¹¹ is H, R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴C(═O)X²R¹⁶, —R¹⁴X²R¹⁶,        —R¹⁴C(═O)X², wherein X² is —O—, —S—, —NH—, —NHS(O₂), —N(R¹⁴)—,        —O—R¹⁴—, —S—R¹⁴—, —S(═O)—R¹⁴—, or —NHR¹⁴—;    -   Wherein R¹² is H, R¹⁴, —O—, —S—, —N—, ═N—, ═NNH—, —OH, —SH,        —NH₂, ═NH, ═NNH₂, —NH(R¹⁴), —OR¹⁴, —C(O)O—, —C(O)OR¹⁶—, —COR¹⁶,        —COOR¹⁴—, C(O)NH—, C(O)NH₂, C(O)NHR¹⁴, —SR¹⁴, —S(═O)R¹⁴,        —P(═O)(OR¹⁶)₂, —OP(═O)(OR¹⁶)₂, —CH₂OP(═O)(OR¹⁶)₂, —SO₂R¹⁶.

Illustrative compounds inside the bracket of formula (II) have preferredstructures below:

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   wherein R²⁰ is H; C₁-C₈ of linear or branched alkyl,        heteroalkyl, or acyl (—C(O)R¹⁷); C₂-C₈ of linear or branched        alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C₃-C₈        linear or branched of aryl, Ar-alkyl, heterocyclic, carbocyclic,        cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl;        carbonate (—C(O)OR¹⁷), carbamate (—C(O)NR¹⁷R¹⁸); or 1-8 carbon        atoms of carboxylate, esters, ether, or amide; or 1˜8 amino        acids; or polyethyleneoxy unit of formula (OCH₂CH₂)_(p) or        (OCH₂CH(CH₃))_(p), wherein p is an integer from 0 to about 1000;        or R²⁰ is absent and the oxygen forms a ketone, or combination        above thereof; wherein R²¹ is H, C₁-C₈ of linear or branched        alkyl;    -   wherein Z³ and Z³ are independently H, OH, NH₂, OR¹⁷, NHR¹⁷,        COOH, COOR¹⁷, C(O)R¹⁷, C(O)NHR¹⁷, C(O)NHNHR¹⁷, C(O)NH₂, R¹⁸,        OCH₂OP(O)(OR¹⁸)₂, OC(O)OP(O)(OR¹⁸)₂, OPO(OR¹⁸)₂, NHPO(OR¹⁸)₂,        OP(O)(OR¹⁸)OP(O)(OR¹⁸)₂, OC(O)R¹⁸, OC(O)NHR¹⁸, OSO₂(OR¹⁸),        O—(C₄-C₁₂₋glycoside), C₁-C₈ of linear or branched alkyl or        heteroalkyl; C₂-C₈ of linear or branched alkenyl, alkynyl,        alkylcycloalkyl, heterocycloalkyl; C₃-C₈ linear or branched of        aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl,        heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate        (—C(O)OR¹⁷), carbamate (—C(O)NR¹⁷R¹⁸); R¹⁷ and R¹⁸ are        independently H, C₁-C₈ linear or branched alkyl or heteroalkyl;        C₂-C₈ of linear or branched alkenyl, alkynyl, alkylcycloalkyl,        heterocycloalkyl; C₃-C₈ linear or branched of aryl, Ar-alkyl,        heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,        alkylcarbonyl, heteroaryl; carbonate (—C(O)OR¹⁷), carbamate        (—C(O)NR¹⁷R¹⁸); R¹⁹ is H, OH, NH₂, OSO₂(OR¹⁸), XCH₂OP(O)(OR¹⁸)₂,        XPO(OR¹⁸)₂, XC(O)OP(O)(OR¹⁸)₂, XC(O)R¹⁸, XC(O)NHR¹⁸, C₁-C₈ alkyl        or carboxylate; C₂-C₈ alkenyl, alkynyl, alkylcycloalkyl,        heterocycloalkyl; C₃˜C₈ aryl or alkylcarbonyl; or pharmaceutical        salts; X is O, S, NH, NHNH, NHR¹⁷, or CH₂; R⁷ is defined the        same above;    -   wherein “        ” is the site that linked to a linker L of Formula (II).

In another embodiment, a conjugate of a cell bindingmolecule-antimitotic agent has the Formula (III):

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   wherein T, L, m, Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹,        R¹², R¹³ and n are defined the same as in Formula (I) and (II);    -   Wherein R⁷ is independently selected from —R¹⁴—, or        —R¹⁴C(═O)X¹R¹⁵— or —R¹⁴X¹R¹⁵—, wherein R¹⁴ and R¹⁵ are        independently linear or branched C₁˜C₈ of alkyl, heteroalkyl;        C₂˜C₈ of alkenyl, alkynyl; C₃˜C₈ of aryl, heterocyclic,        carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl        heteroalkylcycloalkyl, alkylcarbonyl; X¹ is selected from O, S,        S—S, NH, or NR¹⁴.

Illustrative compounds inside the bracket of Formula (III) have thestructures:

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   wherein R²⁰ is H; C₁-C₈ of linear or branched alkyl,        heteroalkyl, or acyl (—C(O)R¹⁷); C₂-C₈ of linear or branched        alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C₃-C₈        linear or branched of aryl, Ar-alkyl, heterocyclic, carbocyclic,        cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl;        carbonate (—C(O)OR¹⁷), carbamate (—C(O)NR¹⁷R¹⁸); or 1-8 carbon        atoms of carboxylate, esters, ether, or amide; or 1˜8 amino        acids; or polyethyleneoxy unit of formula (OCH₂CH₂)_(p) or        (OCH₂CH(CH₃))_(p), wherein p is an integer from 0 to about 1000;        or R²⁰ is absent and the oxygen forms a ketone, or combination        above thereof;    -   wherein R²¹ and R²² are independently H, C₁-C₈ of linear or        branched alkyl;    -   Z³ and Z³ are independently H, OH, NH₂, OR¹⁷, NHR¹⁷, COOH,        COOR¹⁷, C(O)R⁷, C(O)NHR¹⁷, C(O)NHNHR⁷, C(O)NH₂, R¹⁸,        OCH₂OP(O)(OR⁸)₂, OC(O)OP(O)(OR¹⁸)₂, OPO(OR¹⁸)₂, NHPO(OR¹⁸)₂,        OP(O)(OR¹⁸)OP(O)(OR¹⁸)₂, OC(O)R⁸, OC(O)NHR¹⁸, OSO₂(OR¹⁸),        O—(C₄-C₁₂₋glycoside), C₁-C₈ of linear or branched alkyl or        heteroalkyl; C₂-C₈ of linear or branched alkenyl, alkynyl,        alkylcycloalkyl, heterocycloalkyl; C₃-C₈ linear or branched of        aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl,        heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate        (—C(O)OR¹⁷), carbamate (—C(O)NR¹⁷R¹⁸); R¹⁷ and R¹⁸ are        independently H, C₁-C₈ linear or branched alkyl or heteroalkyl;        C₂-C₈ of linear or branched alkenyl, alkynyl, alkylcycloalkyl,        heterocycloalkyl; C₃-C₈ linear or branched of aryl, Ar-alkyl,        heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,        alkylcarbonyl, heteroaryl; carbonate (—C(O)OR¹⁷), carbamate        (—C(O)NR¹⁷R¹⁸); R¹⁹ is H, OH, NH₂, OSO₂(OR¹⁸), XCH₂OP(O)(OR¹⁸)₂,        XPO(OR¹⁸)₂, XC(O)OP(O)(OR¹⁸)₂, XC(O)R¹⁸, XC(O)NHR¹⁸, C₁-C₈ alkyl        or carboxylate; C₂-C₈ alkenyl, alkynyl, alkylcycloalkyl,        heterocycloalkyl; C₃-C₈ aryl or alkylcarbonyl; or pharmaceutical        salts; X, X¹ and X² are independently O, S, NH, NHNH, or CH₂.

In another embodiment, a cell binding molecule-antimitotic agentconjugate has the Formula (IV):

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   wherein T, L, m, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁰, R¹¹, R¹²,        R¹³ and n are defined the same as in Formula (II);    -   Wherein R⁹ is independently H, —O—, —OR¹⁴—, —OC(═O)R¹⁴—,        —OC(═O)NHR¹⁴—, —OC(═O)NR¹⁴R¹⁵—, —OC(═O)R¹⁴SSR¹⁵—,        —OP(═O)(OR¹⁴)O—, wherein R¹⁴, R¹⁵ are independently H, C₁-C₈ of        alkyl, heteroalkyl; C₃-C₈ of aryl, heteroaryl, heterocyclic,        carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl,        heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or        pharmaceutical salts.

Illustrative compounds inside the bracket of Formula (IV) have thestructures:

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   wherein “        ”, R⁷, R²⁰, R²¹, R²², Z², Z³, and X² are defined the same as        above.

In another embodiment, a cell-binding molecule-antimitotic agentconjugate has the formula (V):

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theirisotopes, optical isomers, racemates, diastereomers or enantiomersthereof;

-   -   wherein T, L, m, Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,        R¹², R¹³ and n are defined the same as in Formula (II);    -   Wherein R¹¹ is —R¹⁴—, —R¹⁴C(═O)R¹⁷—, —R¹⁴C(═O)X²R¹⁷—,        —R¹⁴X²R¹⁷—, —R¹⁴C(═O)X²—, wherein R¹⁷ is independently H, OH,        C₁˜C₈ of alkyl; C₂˜C₈ of alkenyl, alkynyl, heteroalkyl; C₃˜C₈ of        aryl, arylene, heterocyclic, carbocyclic, heterocycloalkyl; or        an amino acid, or two amino acid units; X² is —O—, —S—, —NH—,        —NHS(O₂)—, —NHS(O)—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—, —S(═O)—R¹⁴—, or        —NHR¹⁴—; R¹⁴ is H, C₁˜C₈ of alkyl, heteroalkyl; C₂˜C₈ of        alkenyl, alkynyl; C₃˜C₈ of aryl, heterocyclic, carbocyclic,        cycloalkyl, alkylcycloalkyl, heterocycloalkyl,        heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl.

Illustrative compounds inside the bracket of Formula (V) have thestructures:

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   wherein “        ”, R⁷, R²⁰, R²¹, R²², Z², Z³, and X² are defined the same as        above.

In another embodiment, a conjugates of a cell binding-antimitotic agenthave the formula (VI)

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   wherein T, L, m, Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹¹, R¹³        and n are defined the same as in Formula (II);    -   wherein R¹² is independently R¹⁴, —O—, —S—, —NH—, ═N—, ═NNH—,        —N(R¹⁴)—, —OR¹⁴—, C(O)O—, C(O)NH—, C(O)NR¹⁴—, —SR¹⁴—,        —S(═O)R¹⁴—, —NHR¹⁴—, —CH₂OP(═O)(OR¹⁵)—, —P(═O)(OR¹⁵)—,        —OP(═O)(OR¹⁵)O—, —SO₂R¹⁴, R¹⁴, R¹⁵ are independently C₁-C₈ of        alkyl, heteroalkyl; C₂-C₈ of alkenyl, alkynyl; C₃˜C₈ of aryl,        heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,        heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl,        alkylcarbonyl.

Illustrative compounds inside the bracket of Formula (VI) have thestructures:

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   wherein R⁷, R¹⁷, R²⁰, R²¹, Z², Z³, and X² are defined the same        as above.

In another embodiment, the conjugates of the cell-surface bindingmolecule-antimitotic agents have the Formula (VII):

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof,

-   -   Wherein T, L, n, m, Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R¹⁰, R¹¹, and        R¹² are defined the same as in Formula (II);    -   Wherein R¹³ is C₁˜C₁₀ of alkyl, heteroalkyl, alkyl acid, alkyl        amide, alkyl amine, or Ar; Ar refers to a aromatic or hetero        aromatic group, composed of one or several rings, comprising        four to ten carbon, preferentially four to six carbon atoms. The        term of hetero aromatic group refers one or several carbon on        aromatic group, preferentially one, two or three carbon atoms        are replaced by O, N, Si, Se, P or S, preferentially O, S, N.        The term aryl or Ar also refers to a aromatic group, wherein one        or several H atoms are replaced independently by R¹⁸, F, Cl, Br,        I, OR¹⁶, SR¹⁶, NR¹⁶R¹⁸, N═NR¹⁶, N═R¹⁶, NR¹⁶R¹⁸, NO₂, SOR¹⁶R¹⁸,        SO₂R¹⁶, SO₃R¹⁶, OSO₃R¹⁶, PR¹⁶R¹⁸, POR¹⁶R¹⁸, POO₂R¹⁶R¹⁸,        OPO₃R¹⁶R¹⁸, or POO₃R¹⁶R¹⁸ wherein R¹⁶, R¹⁸ are independently H,        C₁˜C₈ of alkyl; C₂˜C₈ of alkenyl, alkynyl, heteroalkyl; C₃˜C₈ of        aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,        heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl,        alkylcarbonyl; or C₄˜C₁₂ glycosides; or pharmaceutical salts.

Illustrative examples of compounds inside the bracket of Formula (VII)have the structures:

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers thereof;

-   -   wherein “        ”, R⁷, R²⁰, Z², Z³, and X² are defined the same as above; X, X¹,        and X³, are independently O, S, NH, NHNH, NHR¹⁷, CH₂ or absent;        P¹ is H, R¹⁷, P(O)(OH)₂, P(O)(X¹R¹⁷)₂, CH₂P(O)(OH)₂,        S(O²)(X¹R¹⁷), C₆H₁₂O₅ (glycoside), (CH₂CH₂O)_(p)R¹⁷, wherein p        is selected from 0-100, and R¹⁷ is defined above; in addition        X¹P¹ can be absent (together is H).

In another embodiment, the synthetic routes to produce the antimitoticagents and their conjugation to a cell-surface receptor bindingmolecules of the present invention are exampled, but not limited to, asshown in FIGS. 1-21 .

In another embodiment, the releasable linker (L) is a chain of atomsselected from C, N, O, S, Si, and P that covalently connects thecell-surface binding ligand (T) to the potent antimitotic agents. Thelinker may have a wide variety of lengths, such as in the range fromabout 2 to about 100 atoms. The atoms used in forming the linker may becombined in all chemically relevant ways, such as forming alkylene,alkenylene, and alkynylene, ethers, polyoxyalkylene, esters, amines,imines, polyamines, hydrazines, hydrazones, amides, ureas,semicarbazides, carbazides, alkoxyamines, alkoxylamines, urethanes,amino acids, acyloxylamines, hydroxamic acids, and many others. Inaddition, it is to be understood that the atoms forming the releasablelinker (L) may be either saturated or unsaturated, or may be radicals,or may be cyclized upon each other to form divalent cyclic structures,including cyclo alkanes, cyclic ethers, cyclic amines, arylenes,heteroarylenes, and the like in the linker.

The term releasable linker refers to a linker that includes at least onebond that can be broken under physiological conditions, such as apH-labile, acid-labile, base-labile, oxidatively labile, metabolicallylabile, biochemically labile, or enzyme-labile bond. It is appreciatedthat such physiological conditions resulting in bond breaking do notnecessarily include a biological or metabolic process, and instead mayinclude a standard chemical reaction, such as a hydrolysis orsubstitution reaction, for example, an endosome having a lower pH thancytosolic pH, and/or disulfide bond exchange reaction with aintracellular thiol, such as the amillimolar range of abundant ofglutathione inside the malignant cells.

The releasable linker L of conjugates may have the formula:—Ww-(Aa)r-Vv- wherein: —W— is a Stretcher unit; w is 0 or 1; each -Aa-is independently an Amino Acid unit; r is independently an integerranging from 0 to 12; —V— is a Spacer unit; and v is 0, 1 or 2. TheStretcher unit (—W—), when present, links a targeted binding molecularunit (T) to an amino acid unit (-Aa-), or links V when an Aa is notpresent. The Stretcher unit W may independently contain aself-immolative spacer, peptidyl units, a hydrazone bond, disulfide orthiolether bonds. In this regard a binding molecular (T) has afunctional group that can form a bond with a functional group of aStretcher. Useful functional groups that can be present on a bindingmolecular, either naturally or via chemical manipulation include, butare not limited to, sulfhydryl (—SH), amino, hydroxyl, carbonyl, theanomeric hydroxyl group of a carbohydrate, and carboxyl. Preferredfunctional groups are sulfhydryl, carboxy and amino. Sulfhydryl groupscan be generated by reduction of an intramolecular disulfide bond of aLigand. Alternatively, sulfhydryl groups can be generated by reaction ofan amino group of a lysine moiety of a binding molecular using2-iminothiolane (Traut's reagent) or thiolactone or another sulfhydrylgenerating reagent, such as modifies T with a disulfide bond linker, ora thiol ester following by reduction or hydrolysis respectively.

Illustrative examples of W linked to T have the structures:

wherein R²⁰ and R²¹ are independently selected from —C₁˜C₉ alkylene-,—C₁˜C₇ carbocyclo-, —O—(C₁˜C₈ alkyl)-, -arylene-, —C₁˜C₉alkylene-arylene-, -arylene, —C₁˜C₉ alkylene-, —C₁˜C₉ alkylene-(C₁˜C₈carbocyclo)-, —(C₃˜C₇ carbocyclo)-C₁˜C₉ alkylene-, —C₃˜C₈ heterocyclo-,—C₁˜C₁₀ alkylene-(C₃˜C₈ heterocyclo)-, —(C₃˜C₈ heterocyclo)-C₁˜C₉alkylene-, —(CH₂CH₂O)_(k)—, —(CH(CH₃)CH₂O)_(k)—, and—(CH₂CH₂O)_(k)—CH₂—; k is an integer ranging from 1-20; R′ and R″ areindependently H or CH₃.

In another embodiment, conjugation of W to T covalently as illustratedabove can be via various chemical reactions.

Examples of the Formation of Amide Linkages:

Wherein the Stretcher unit contains a reactive site of E, which can forman amide bond with a primary or secondary amino group of a Ligand.Example of the reactive E, includes, but is not limited to, such ashydroxysuccinimidyl esters (NHS, Sulfo-NHS, etc), 4-nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl (includessulfo-tetrafluorophenyl) esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates and isothiocyanates.

Examples of Thiol Ether or Disulfide Bond Linkages:

Wherein the Stretcher unit contains a sulfhydryl reactive site, whichcan form a thiol ether or disulfide bond with a thiol group which isgenerated by reduction of an intramolecular disulfide bond of thebinding ligand T, or generated by a chemical modification on the bindingligand T.

In yet another aspect of the invention, the reactive group of theStretcher contains a reactive site that is reactive to an aldehyde(—CHO) or a ketone (—C(═O)R) group that can be chemically modified on abinding molecular T. For example, a carbohydrate on a binding molecularT can be mildly oxidized using a reagent such as sodium periodate togenerate an aldehyde or a ketone (—C(═O)R) group; or an amine on anamino acid at the N-termini of antibodies (or proteins or peptides) canreact with pyridoxal 5′-phosphate (PLP) in a buffer solution tointroduce ketone groups (Scheck & Francis, ACS Chem. Biol. 2007, 2,247-251). The resulting (—C═O) unit can be condensed with a Stretcherthat contains a functionality such as a hydrazide, an oxime, a primaryor secondary amine, a hydrazine, a thiosemicarbazone, a hydrazinecarboxylate, and an arylhydrazide.

Examples of the Conjugation of the Hydrazone, or the Oxime or ImineLinkages:

wherein R²⁰ and R²¹ are described above, R²⁵ is an organic substituentof an amino acid.

In another aspect of the invention, the Stretchers (which may contain aspacer V and/or an amino acid) can be linked to the binding molecules(T), followed by conjugation of a potent antimitotic agent to thebinding molecule-stretcher moiety in an aqueous buffered solution.Examples of these kinds of two-step conjugations (a drug linked to R¹⁶is omitted here):

wherein E includes, but is not limited to, such as hydroxysuccinimidylesters (NHS, Sulfo-NHS, etc), 4-nitrophenyl esters, pentafluorophenylesters, tetrafluorophenyl (includes sulfo-tetrafluorophenyl) esters,anhydrides, acid chlorides, sulfonyl chlorides, isocyanates andisothiocyanates. R′ and R″ are independently H or CH₃; R²⁰, R¹⁶ and Arare defined in various embodiment throughout this inventions; R²⁶ is H,or F, or NO₂ independently; J is F, Cl, Br, I, tosylate (TsO) ormesylate (MsO) independently and wherein

-R₁₆ bears at least one antimitotic agent/drug (Drug)_(n)

R¹⁶.

In another aspect of the invention, the Stretchers can be linked to apotent antimitotic agent first, followed by conjugation of the bindingmolecules (T) in an aqueous pH 3˜10 (preferably pH 5˜8.5) bufferedsolution containing up to 50% of organic cosolvents. Examples of thesekinds of two-step conjugations:

wherein E includes, but is not limited to, such as hydroxysuccinimidylesters (NHS, Sulfo-NHS, etc), 4-nitrophenyl esters, pentafluorophenylesters, tetrafluorophenyl (includes sulfo-tetrafluorophenyl) esters,anhydrides, acid chlorides, sulfonyl chlorides, isocyanates andisothiocyanates. R′ and R″ are independently H or CH₃; R¹⁶, R²⁰ and Arare defined in various embodiment throughout this inventions; R²⁶ is H,or F, or NO₂ independently; J is F, Cl, Br, I, tosylate (TsO) ormesylate (MsO) independently and wherein

bears at least one antimitotic agent/drug.

The Amino Acid unit (-Aa-), when present, links the Stretcher unit tothe Spacer unit if the Spacer unit is present, links the Stretcher unitto the antimitotic agent unit if the Spacer unit is absent, and linksthe binding molecule (T) unit to the antimitotic agent unit if theStretcher unit and Spacer unit are absent. -(Aa)r- is a natural orunnatural amino acid, dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit, and r is an integer ranging from 0to 12. The term amino acid as used herein refers generally toaminoalkylcarboxylate, where the alkyl radical is optionallysubstituted, such as with alkyl, acyl, hydroxy alkyl, sulfhydrylalkyl,aminoalkyl, carboxyalkyl, and the like, The structures of the naturaland unnatural amino acids and peptides are described in the book: G. C.Barrett and D. T. Elmore, “Amino Acid and Peptide”, Cambridge UniversityPress, 2004. In addition, amino acid refers to beta, gamma, and longeramino acids with intra chain containing methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like.More preferably the amino acid is selected from asparagine, asparticacid, cysteine, glycine, glutamic acid, lysine, glutamine, arginine,serine, ornithine, threonine, and the like.

The Amino Acid unit of the Invention can be enzymatically cleaved by oneor more enzymes, including a tumor-associated protease, to liberate theantimitotic agent, which in one embodiment is protonated in vivo uponrelease to provide an antimitotic agent.

The Spacer unit (—V—), when present, links an Amino Acid unit to theantimitotic agent when an Amino Acid unit is present. Alternately, theSpacer unit links the Stretcher unit to antimitotic agent when the AminoAcid unit is absent. The Spacer unit also links antimitotic agent to thebinding molecule (T) when both the Amino Acid unit and Stretcher unitare absent. The spacer linkers may contain function groups thatsubstantially increase the water solubility, biological transport,preferential renal clearance, uptake, absorption, biodistribution,and/or bioavailability of the conjugate are described herein. Spacerunits are of two general types: self-immolative and non-self-immolative.A non-self-immolative Spacer unit is one in which part or all of theSpacer unit remains bound to antimitotic agent after cleavage,particularly enzymatic, of an Amino Acid unit from the antimitoticagent-Linker- binding molecule conjugate or the antimitotic agent-LinkerCompound. The self-immolative unit includes aromatic compounds that areelectronically similar to the para-aminobenzyl-carbamoyl (PAB) groups,2-aminoimidazol-5-methanol derivatives, heterocyclic PAB analogs,beta-glucuronide, and ortho or para-aminobenzylacetals; or one of thefollowing structures:

wherein the (*) atom is the point of attachment of additional spacer orreleasable linker units, the antimitotic agent, and/or the bindingmolecule (T); X, Y and Z³ are independently NH, O, or S; Z² is H, NH, Oor S independently. v is 0 or 1; Q is independently H, OH, C₁˜C₆ alkyl,(OCH₂CH₂)_(n)F, Cl, Br, I, OR¹⁷, or SR¹⁷, NR¹⁷R¹⁸, N═NR¹⁷, N═R¹⁷,NR¹⁷R¹⁸, NO₂, SOR¹⁷R¹⁸, SO₂R¹⁷, SO₃R¹⁷, OSO₃R¹⁷, PR¹⁷R¹⁸, POR¹⁷R¹⁸,PO₂R¹⁷R¹⁸, OPO(OR¹⁷)(OR¹⁸), or OCH₂PO(OR¹⁷(OR¹⁸) wherein R¹⁷, R¹⁸ areindependently H, C₁˜C₈ of alkyl; C₂˜C₈ of alkenyl, alkynyl, heteroalkyl;C₃˜C₈ of aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl,heteroaralkyl, alkylcarbonyl; or pharmaceutical cation salts

Examples of the Non-Self-Immolative Spacer Linkers:

or L- or D-, natural or unnatural peptides containing 1-20 the same ordifferent amino acids;

Wherein the “*” and “

” atom are the point of attachment of additional spacer or releasablelinkers, the antimitotic agents, and/or the binding molecules; m is1˜10; n is 1˜20; X₂, X₃, X₄, X₅, or X₆, are independently selected fromNH; NHNH; N(R₁₂); N(R₁₂)N(R_(12′)); O; S; C₁-C₆ of alkyl; C₂-C₆ ofheteroalkyl, alkylcycloalkyl, heterocycloalkyl; C₃-C₈ of aryl, Ar-alkyl,heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,alkylcarbonyl, heteroaryl; CH₂OR₁₂, CH₂SR₁₂, CH₂NHR₁₂, or 1˜8 aminoacids; wherein R₁₂ and R_(12′) are independently H; C₁-C₈ of alkyl;C₂-C₈ of hetero-alkyl, alkylcycloalkyl, heterocycloalkyl; C₃-C₈ of aryl,Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,alkylcarbonyl, heteroaryl; or 1-8 carbon atoms of esters, ether, oramide; or polyethyleneoxy unit of formula (OCH₂CH₂)_(p) or(OCH₂CH(CH₃))_(p), wherein p is an integer from 0 to about 1000, orcombination above thereof.

A releasable component of the linker L that at least one bond in L canbe broken under physiological conditions: a pH-labile, acid-labile,base-labile, oxidatively labile, metabolically labile, biochemicallylabile or enzyme-labile bond, which having one of the followingstructures:

—(CR₁₅R₁₆)_(m)(Aa)r(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(t)—,—(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(Aa)r(OCH₂CH₂)_(t)—,-(Aa)_(r)-(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(t)—,—(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(r)(Aa)_(t)-,—(CR₁₅R₁₆)_(m)(CR₁₇═CR₁₈)(CR₁₉R₂₀)_(n)(Aa)_(t)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(NR₁₁CO)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(Aa)_(t)(NR₂₁CO)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(OCO)(Aa)_(t)-(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(OCNR₁₇)(Aa)_(t)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)—(CO)(Aa)_(t)-(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(NR₂₁CO)(Aa)_(t)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)—(OCO)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(OCNR₁₇)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(CO)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)-phenyl-CO(Aa)_(t)-(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(m)-furyl-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(m)-oxazolyl-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(m)-thiazolyl-CO(Aa)_(t)(CCR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(t)-thienyl-CO(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(t)-imidazolyl-CO—(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(t)-morpholino-CO(Aa)_(t)-(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(t)-piperazino-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(t)—N-methylpiperazin-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(m)-(Aa)_(t)phenyl-, —(CR₁₅R₁₆)_(m)-(Aa)_(t)furyl-,—(CR₁₅R₁₆)_(m)-oxazolyl(Aa)_(t)-, —(CR₁₅R₁₆)_(m)-thiazolyl(Aa)_(t)-,—(CR₁₅R₁₆)_(m)-thienyl-(Aa)_(t)-, —(CR₁₅R₁₆)_(m)-imidazolyl(Aa)_(t)-,—(CR₁₅R₁₆)_(m)-morpholino-(Aa)_(t)-,—(CR₁₅R₁₆)_(m)-piperazino-(Aa)_(t)-,—(CR₁₅R₁₆)_(m)—N-methylpiperazino-(Aa)_(t)-,—K(CR₁₅R₁₆)_(m)(Aa)r(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(t)—,—K(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(Aa)_(r)(OCH₂CH₂)_(t)—,—K(Aa)_(r)-(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(t)—,—K(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(r)(Aa)_(t)-,—K(CR₁₅R₁₆)_(m)—(CR₁₇═CR₁₈)(CR₁₉R₂₀)_(n)(Aa)_(t)(OCH₂CH₂)_(r),—K(CR₁₅R₁₆)_(m)(NR₁₁CO)(Aa)_(t)-(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)(Aa)_(t)(NR₂₁CO)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)—(OCO)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂H₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)(OCNR₁₇)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)(CO)(Aa)_(t)-(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)(NR₂₁CO)(Aa)_(t)-(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)—(OCO)(Aa)_(t)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)—(OCNR₁₇)(Aa)_(t)-(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K—(CR₁₅R₁₆)_(m)(CO)(Aa)_(t)(CR₁₉R₂₀)_(n)(OCH₂C₂)_(r)—,—K(CR₁₅R₁₆)_(m)-phenyl-CO(AA)_(t)(CR₁₇R₁₈)_(n)—,—K—(CR₁₅R₁₆)_(m)-furyl-CO(AA)_(t)(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(m)-oxazolyl-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(m)-thiazolyl-CO(Aa)_(t)-(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(t)-thienyl-CO(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(t)imidazolyl-CO—(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₅)_(t)morpholino-CO(Aa)_(t)-(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(t)-piperazino-CO(Aa)_(t)-(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(t)—N-methylpiperazin-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(m)-(Aa)_(t)phenyl, —K—(CR₁₅R₁₆)_(m)-(Aa)_(t)furyl-,—K(CR₁₅R₁₆)_(m)-oxazolyl-(Aa)_(t)-, —K(CR₁₅R₁₆)_(m)-thiazolyl(Aa)_(t)-,—K(CR₁₅R₁₆)_(m)-thienyl-(Aa)_(t)-, —K(CR₁₅R₁₆)_(m)-imidazolyl(Aa)_(t)-,—K(CR₁₅R₁₆)_(m)-morpholino(Aa)_(t)-, —K(CR₁₅R₁₆)_(m)piperazino(Aa)_(t)G,—K(CR₁₅R₁₆)_(m)—N-methyl-piperazino(Aa)_(t)-; wherein m, Aa, m, n, R₁₃,R₁₄, and R₁₅ are described above; t and r here are 0-100 independently;R₁₆, R₁₇, R₁₈, R₁₉, and R₂₀ are independently chosen from H; halide;C₁˜C₈ of alkyl or heteroalkyl, C₂˜C₈ of aryl, alkenyl, alkynyl, ether,ester, amine or amide, C₃-C₈ of aryl, which optionally substituted byone or more halide, CN, NR₁₂R_(12′), CF₃, OR₁₂, Aryl, heterocycle,S(O)R₁₂, SO₂R₁₂, —CO₂H, —SO₃H, —OR₁₂, —COO₂R₁₂, —CONR₁₂, —PO₂R₁₂R₁₃,—PO₃H or P(O)R₁₂R_(12′)R₁₃; K is NR₁₂, —SS—, —C(═O)—, —C(═O)NH—,—C(═O)O—, —C═NH—O—, —C═N—NH—, —C(═O)NH—NH—, O, S, Se, B, Het(heterocyclic or heteroaromatic ring having C₃-C₁₂); or peptidescontaining the same or different 1-20 amino acids.

The binding molecule (T) may be of any kind presently known, or thatbecome known, molecule that binds to, complexes with or reacts with amoiety of a cell population sought to be therapeutically or otherwisebiologically modified. The binding molecule unit acts to deliver theantimitotic agents to the particular target cell population with whichthe binding molecule (T) reacts.

The cell-binding agents, T include, but are not limited to, largemolecular weight proteins such as, for example, full-length antibodies(polyclonal and monoclonal antibodies); single chain antibodies;fragments of antibodies such as Fab, Fab′, F(ab′)₂, F_(v), [Parham, J.Immunol. 131, 2895-2902 (1983)], fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-bindingfragments of any of the above which immuno-specifically bind to cancercell antigens, viral antigens or microbial antigens; interferons (suchas type I, II, III); peptides; lymphokines such as IL-2, IL-3, IL-4,IL-6, GM-CSF, interferon-gamma (IFN-γ); hormones such as insulin, TRH(thyrotropin releasing hormones), MSH (melanocyte-stimulating hormone),steroid hormones, such as androgens and estrogens,melanocyte-stimulating hormone (MSH); growth factors andcolony-stimulating factors such as epidermal growth factors (EGF),granulocyte-macrophage colony-stimulating factor (GM-CSF), transforminggrowth factors (TGF), such as TGFα, TGFβ, insulin and insulin likegrowth factors (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF [Burgess,Immunology Today, 5, 155-158 (1984)]; vaccinia growth factors (VGF);fibroblast growth factors (FGFs); smaller molecular weight proteins,poly-peptide, peptides and peptide hormones, such as bombesin, gastrin,gastrin-releasing peptide; platelet-derived growth factors; interleukinand cytokines, such as interleukin-2 (IL-2), interleukin-6 (IL-6),leukemia inhibitory factors, granulocyte-macrophage colony-stimulatingfactor (GM-CSF); vitamins, such as folate; apoproteins andglycoproteins, such as transferrin {O'Keefe et al, 260 J. Biol. Chem.932-937 (1985)}; sugar-binding proteins or lipoproteins, such aslectins; cell nutrient-transport molecules; and small molecularinhibitors, such as prostate-specific membrane antigen (PSMA) inhibitorsand small molecular tyrosine kinase inhibitors (TKI), non-peptides orany other cell binding molecule or substance, such as bioactive polymers(Dhar, et al, Proc. Natl. Acad. Sci. 2008, 105, 17356-61); dendrimers(Lee, et al, Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc.Natl. Acad. Sci. 2009, 106, 685-90); nanoparticles (Liong, et al, ACSNano, 2008, 19, 1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7;Javier, et al, Bioconjugate Chem. 2008, 19, 1309-12); liposomes(Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9); viral capsides(Flenniken, et al, Viruses Nanotechnol. 2009, 327, 71-93). In generalmonoclonal antibodies are preferred as a cell-surface binding agent ifan appropriate one is available.

Preferably, T is selected from the group consisting of an antibody, asingle chain antibody, an antibody fragment that binds to a target cell,a monoclonal antibody, a single chain monoclonal antibody, a monoclonalantibody fragment that binds to the target cell, a chimeric antibody, achimeric antibody fragment that binds to the target cell, a domainantibody, a domain antibody fragment that binds to the target cell, anadnectin that mimics antibody, DARPins, a lymphokine, a hormone, avitamin, a growth factor, a colony stimulating factor, anutrient-transport molecule (a transferrin), and/or a cell-bindingpeptide, protein, or small molecule attached or coated on an albumin, apolymer, a dendrimer, a liposome, a nanoparticle, a vesicle, or on a(viral) capsid.

In further preferably, the cell binding agent/molecule, T is capable oftargeting against a tumor cell, a virus infected cell, a microorganisminfected cell, a parasite infected cell, an autoimmune disease cell, anactivated tumor cells, a myeloid cell, an activated T-cell, an affectingB cell, or a melanocyte, or any disease cells expressing any one of thefollowing antigens or receptors: CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2,CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10,CD11a, CD11b, CD11c, CD11d, CD12w, CD13, CD14, CD15, CD16, CD16a, CD16b,CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28,CD29, CD30, CD31, CD32, CD32a, CD32b, CD33, CD34, CD35, CD36, CD37,CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44,CD45, CD46, CD47, CD48, CD49b, CD49c, CD49c, CD49d, CD49f, CD50, CD51,CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD60a, CD60b,CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a,CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72,CD73, CD74, CD75, CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b, CD80,CD81, CD82, CD83, CD84, CD85, CD85a, CD85b, CD85c, CD85d, CD85e, CD85f,CD85g, CD85g, CD85i, CD85j, CD85k, CD85m, CD86, CD87, CD88, CD89, CD90,CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101,CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109,CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119,CD120, CD120a, CD120b, CD121, CD121a, CD121b, CD122, CD123, CD123a,CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD131, CD132, CD133,CD134, CD135, CD136, CD137, CD138, CD139, CD140, CD140a, CD140b, CD141,CD142, CD143, CD144, CD145, CDw145, CD146, CD147, CD148, CD149, CD150,CD151, CD152, CD153, CD154, CD155, CD156, CD156a, CD156b, CD156c,CD156d, CD157, CD158, CD158a, CD158b1, CD158b2, CD158c, CD158d, CD158e1,CD158e2, CD158f2, CD158 g, CD158 h, CD158i, CD158j, CD158k, CD159,CD159a, CD159b, CD159c, CD160, CD161, CD162, CD163, CD164, CD165, CD166,CD167, CD167a, CD167b, CD168, CD169, CD170, CD171, CD172, CD172a,CD172b, CD172 g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178,CD179, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184, CD185, CD186,CDw186, CD187, CD188, CD189, CD190, CD191, CD192, CD193, CD194, CD195,CD196, CD197, CD198, CD199, CDw198, CDw199, CD200, CD201, CD202,CD202(a,b), CD203, CD203c, CD204, CD205, CD206, CD207, CD208, CD209,CD210, CDw210a, CDw210b, CD211, CD212, CD213, CD213a₁, CD213a₂, CD214,CD215, CD216, CD217, CD218, CD218a, CD218, CD21b9, CD220, CD221, CD222,CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232,CD233, CD234, CD235, CD235a, CD235b, CD236, CD237, CD238, CD239, CD240,CD240ce, CD240d, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248,CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256, CD257, CD258,CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268,CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278,CD279, CD281, CD282, CD283, CD284, CD285, CD286, CD287, CD288, CD289,CD290, CD291, CD292, CD293, CD294, CD295, CD296, CD297, CD298, CD299,CD300, CD300a, CD300b, CD300c, CD301, CD302, CD303, CD304, CD305, CD306,CD307, CD307a, CD307b, CD307c, CD307d, CD307e, CD307f, CD308, CD309,CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319,CD320, CD321, CD322, CD323, CD324, CD325, CD326, CD327, CD328, CD329,CD330, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339,CD340, CD341, CD342, CD343, CD344, CD345, CD346, CD347, CD348, CD349,CD350, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358, CD359,CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369,CD370, CD371, CD372, CD373, CD374, CD375, CD376, CD377, CD378, CD379,CD381, CD382, CD383, CD384, CD385, CD386, CD387, CD388, CD389, CRIPTO,CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, 4-1BB, APO2, ASLG659,BMPR1B, 4-1BB, 5AC, 5T4 (Trophoblastic glycoprotein, TPBG, 5T4,Wnt-Activated Inhibitory Factor 1 or WAIF 1), Adenocarcinoma antigen,AGS-5, AGS-22M6, Activin receptor-like kinase 1, AFP, AKAP-4, ALK, Alphaintegrin, Alpha v beta6, Amino-peptidase N, Amyloid beta, Androgenreceptor, Angiopoietin 2, Angiopoietin 3, Annexin A1, Anthrax toxinprotective antigen, Anti-transferrin receptor, AOC3 (VAP-1), B7-H3,Bacillus anthracis anthrax, BAFF (B-cell activating factor), BCMA,B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125(carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase9), CALLA, CanAg, Canis lupus familiaris IL31, Carbonic anhydrase IX,Cardiac myosin, CCL11(C—C motif chemokine 11), CCR4 (C—C chemokinereceptor type 4), CCR5, CD3E (epsilon), CEA (Carcinoembryonic antigen),CEACAM3, CEACAM5 (carcino-embryonic antigen), CFD (Factor D), Ch4D5,Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), CLDN18.1 (Claudin-18.1),CLDN18.2 (Claudin-18.2), Clumping factor A, cMet, CRIPTO, FCSF1R (Colonystimulating factor 1 receptor), CSF2 (colony stimulating factor 2,Granulocyte-macrophage colony-stimulating factor (GM-CSF)), CSP4, CTLA4(cytotoxic T-lymphocyte-associated protein 4), CTAA16.88 tumor antigen,CXCR4, C—X—C chemokine receptor type 4, cyclic ADP ribose hydrolase,Cyclin B1, CYP1B1, Cytomegalovirus, Cytomegalovirus glycoprotein B,Dabigatran, DLL3 (delta-like-ligand 3), DLL4 (delta-like-ligand 4), DPP4(Dipeptidyl-peptidase 4), DR5 (Death receptor 5), E. coli shiga toxintype-1, E. coli shiga toxin type-2, ED-B, EGFL7 (EGF-likedomain-containing protein 7), EGFR, EGFRII, EGFRvIII, Endoglin,Endothelin B receptor, Endotoxin, EpCAM (epithelial cell adhesionmolecule), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2),ERBB3, ERG (TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP(Fibroblast activation protein alpha), fibroblast surface antigen,FCGR1, alpha-Fetoprotein, Fibrin II, beta chain, Fibronectin extradomain-B, FOLR (folate receptor), Folate receptor alpha, Folatehydrolase, Fos-related antigen 1F protein of respiratory syncytialvirus, Frizzled receptor, Fucosyl GM1, GD2 ganglioside, G-28 (a cellsurface antigen glycolipid), GD3 idiotype, GloboH, Glypican 3,N-glycolylneuraminic acid, GM3, GMCSF receptor α-chain, Growthdifferentiation factor, GP100, GPNMB (Trans-membrane glycoprotein NMB),GUCY2C (Guanylate cyclase 2C, guanylyl cyclase C(GC-C), intestinalGuanylate cyclase, Guanylate cyclase-C receptor, Heat-stable enterotoxinreceptor (hSTAR)), Heat shock proteins, Hemagglutinin, Hepatitis Bsurface antigen, Hepatitis B virus, HER1 (human epidermal growth factorreceptor 1), HER2, HER2/neu, HER3 (ERBB-3), IgG4, HGF/SF (Hepatocytegrowth factor/scatter factor), HHGFR, HIV-1, Histone complex, HLA-DR(human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, Human chorionicgonadotropin, HNGF, Human scatter factor receptor kinase, HPV E6/E7,Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), Idiotype,IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ,Influenza hemagglutinin, IgE, IgE Fc region, IGHE, interleukins(comprising IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-17A, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-27, or IL-28), IL31RA, ILGF2 (Insulin-likegrowth factor 2), Integrins (α4, α_(IIb)β₃, αvβ3, α₄β₇, α5β1, α6β4,α7β7, α11β3, α5β5, αvβ5), Interferon gamma-induced protein, ITGA2,ITGB2, KIR2D, Kappa Ig, LCK, Le, Legumain, Lewis-Y antigen, LFA-1(Lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1,Lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2,MAGE-3, MAGE A1, MAGE A3, MAGE 4, MART1, MCP-1, MIF (Macrophagemigration inhibitory factor, or glycosylation-inhibiting factor (GIF)),MS4A1 (membrane-spanning 4-domains subfamily A member 1), MSLN(mesothelin), MUC1 (Mucin 1, cell surface associated (MUC1) orpolymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1(monocyte chemotactic protein 1), MelanA/MART1, ML-IAP, MPG, MS4A1(membrane-spanning 4-domains subfamily A), MYCN, Myelin-associatedglycoprotein, Myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen),Nectin-4 (ASG-22ME), NGF, Neural apoptosis-regulated proteinase 1,NOGO-A, Notch receptor, Nucleolin, Neu oncogene product, NY-BR-1,NY-ESO-1, OX-40, OxLDL (Oxidized low-density lipoprotein), OY-TES1, P21,p53 nonmutant, P97, Page4, PAP, Paratope of anti-(N-glycolylneuraminicacid), PAX3, PAX5, PCSK9, PDCD1 (PD-1, Programmed cell death protein 1),PDGF-Ra (Alpha-type platelet-derived growth factor receptor), PDGFR-13,PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase,Platelet-derived growth factor receptor beta, Phosphate-sodiumco-transporter, PMEL 17, Polysialic acid, Proteinase3 (PR1), Prostaticcarcinoma, PS (Phosphatidylserine), Prostatic carcinoma cells,Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD(Rh polypeptide 1 (RhPI)), Rhesus factor, RANKL, RhoC, Ras mutant, RGS5,ROBO4, Respiratory syncytial virus, RON, ROR1, Sarcoma translocationbreakpoints, SART3, Sclerostin, SLAMF7 (SLAM family member 7), SelectinP, SDC1 (Syndecan 1), sLe(a), Somatomedin C, SIP(Sphingosine-1-phosphate), Somatostatin, Sperm protein 17, SSX2, STEAP1(six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn,TAG-72 (tumor associated glycoprotein 72), Survivin, T-cell receptor, Tcell transmembrane protein, TEM1 (Tumor endothelial marker 1), TENB2,Tenascin C (TN-C), TGF-α, TGF-β (Transforming growth factor beta),TGF-β1, TGF-β2 (Transforming growth factor-beta 2), Tie (CD202b), Tie2,TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosisfactor receptor superfamily member 10B), TNFRSF-13B (tumor necrosisfactor receptor superfamily member 13B), TPBG (trophoblastglycoprotein), TRAIL-R₁ (Tumor necrosis apoptosis Inducing ligandReceptor 1), TRAILR2 (Death receptor 5 (DR5)), tumor-associated calciumsignal transducer 2, tumor specific glycosylation of MUC1, TWEAKreceptor, TYRP1 (glycoprotein 75), TRP-1 (Trop1), TRP-2 (Trop2),Tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF-2, VEGFR-1, VEGFR2, or vimentin,WT1, XAGE 1, or cells expressing any insulin growth factor receptors, orany epidermal growth factor receptors.

In the process of the conjugation, prior to conjugating with theantimitotic agents of this invention, the cell-binding molecules can bemodified through attachment of a more specific peptide, a protein, or adrug, or the other functional molecules with a heterobifunctional crosslinker such as with linkers of Amine-to-Nonselective (succinimidyl(NHS)-diazirine (SDA), NHS ester/Azide), Amine-to-Sulfhydryl (NHSester/maleimide, NHS ester/pyridyldithiol, NH S esters/haloacetyl),Sulfhydryl-to-Carbohydrate (Maleimide/Hydrazide,Pyridyldithiol/Hydrazide), Hydroxyl-to-Sulfhydryl(Isocyanate/Maleimide), Amine-to-DNA (NHS ester/Psoralen),Amine-to-Carboxyl (Carbodiimide).

In the SDA linkage modification, the NHS ester of a SDA linker reactswith primary an amine group of a binding molecule backbone in pH 6˜9buffer to form a stable amide bond upon release of NHS, Thenphotoactivation of the diarzirine with long-wave UV light (330-370 nm)creates a reactive carbene intermediate that can react with an aminegroup of a more specific peptide or a protein or the other functionalmolecule. The order of these two steps can be different as this: anamine group of a functional molecule reacts with a SDA linker firstfollowing by photoactive reaction of a binding molecule with long-waveUV light (330-370 nm). The SDA crosslinkers can be cleavable (with adisulfide bond inside such as SDAD linker).

In the NHS ester/Azide linkage modification, the NHS ester of the linkerreacts with primary an amine group of a binding molecule backbone in pH6˜9 buffer to form a stable amide. Then an alkynyl group on a morespecific peptide or a protein or the other functional molecule reacts tothe azide on the other side of the linker via Azide-Alkyne HuisgenCycloaddition to form a 1,2,3-triazole linkage (click chemistry). Also,the NHS ester of the linker reacts with primary an amine group of afunctional molecule in pH 6˜9 buffer to form a stable amide. Then nalkynyl group being linked on a binding molecule reacts to the azide onthe other side of the linker via 5 Azide-Alkyne Huisgen Cycloaddition toform a 1,2,3-triazole linkage.

In the Amine-to-Sulfhydryl linkage modification, the NHS ester of thelinker reacts with a primary amine group of a binding molecule backbonein pH 6˜9 buffer to form a stable amide bond. Then a sulfhydryl on amore specific peptide or a protein or the other functional moleculereacts to the maleimide, or pyridyldithiol, or haloacetyl on the otherside of the Amine-tosulfhydryl linker at pH 4.5˜8.5 to form a thioetheror a disulfide bond. The conjugation with the Amine-to-Sulfhydryl linkercan be in different orders. For instance, an amine group of a functionalmolecule can be reacted with the linker to form an amide bond first,following by reaction with a sulfhydryl on a binding molecule. Also asulfhydryl group of a functional molecule can be reacted with the linkerto form a thioether or a disulfide bond at pH-4.5˜7 first, following byreaction with an amine group on a binding molecule at pH 6˜9 to form anamide bond.

In the Sulfhydryl-to-Carbohydrate linkage modification, the sulfhydrylgroup of a binding molecule can be reacted with the maleimide or thepyridyldithiol on the linker to form a thioether or a disulfide bond atpH 4.5˜8 first, Then a carbonyl (aldehyde/ketone) group on a functionalmolecule reacts with the hydrazide to form an hydrazone bond. Also thesulfhydryl group on a functional molecule can react with the linker toform a thioether or a disulfide bond at pH 4.5˜8 first, following byreaction with a carbohydrate, or an oxidized carbohydrate, or ancarbonyl (aldehyde/ketone) group on a binding molecule form an hydrazonebond.

In the Hydroxyl-to-Sulfhydryl linkage modification, the sulfhydryl groupof a binding molecule can be reacted with the maleimide or thepyridyldithiol on the linker to form a thioether or a disulfide bond atpH 6˜8 first, Then a hydroxy group on a functional molecule reacts withthe isocyanate on the linker to form a carbamate bond at pH 8˜9. Alsothe sulfhydryl group on a functional molecule can react with the linkerto form a thioether or a disulfide bond at pH 6˜8 first, following byreaction with a hydroxy on a binding molecule form a carbamate bond atpH 8˜9.

In yet another aspect of the invention, the production of antibodiesused in the present invention involves in vivo or in vitro procedures orcombinations thereof. Methods for producing polyclonal anti-receptorpeptide antibodies are well-known in the art, such as in U.S. Pat. No.4,493,795 (to Nestor et al). A monoclonal antibody is typically made byfusing myeloma cells with the spleen cells from a mouse that has beenimmunized with the desired antigen (Köhler, G.; Milstein, C. (1975).Nature 256: 495-497). The detailed procedures are described in“Antibodies—A Laboratory Manual”, Harlow and Lane, eds., Cold SpringHarbor Laboratory Press, New York (1988), which is incorporated hereinby reference. Particularly monoclonal antibodies are produced byimmunizing mice, rats, hamsters or any other mammal with the antigen ofinterest such as the intact target cell, antigens isolated from thetarget cell, whole virus, attenuated whole virus, and viral proteins.Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 6000. Fused hybrids are selected by their sensitivity toHAT (hypoxanthine-aminopterin-thymine). Hybridomas producing amonoclonal antibody useful in practicing this invention are identifiedby their ability to immunoreact specified receptors or inhibit receptoractivity on target cells.

A monoclonal antibody used in the present invention can be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques, such as using protein-A affinitychromatography; anion, cation, hydrophobic, or size exclusivechromatographies (particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography); centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media. An exemplary synthetic medium is Dulbecco's minimalessential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959))supplemented with 4.5 gm/1 glucose, 20 mm glutamine, 20% fetal calfserum and with an anti-foaming agent, such aspolyoxyethylene-polyoxypropylene block copolymer.

In addition, antibody-producing cell lines can also be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with an oncovirus, suchas Epstein-Barr virus (EBV, also called human herpesvirus 4 (HHV-4)) orKaposi's sarcoma-associated herpesvirus (KSHV). See, U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890. A monoclonal antibody may also beproduced via an anti-receptor peptide or peptides containing thecarboxyl terminal as described well-known in the art. See Niman et al.,Proc. Natl. Acad. Sci. USA, 80: 4949-4953 (1983); Geysen et al., Proc.Natl. Acad. Sci. USA, 82: 178-182 (1985); Lei et al. Biochemistry34(20): 6675-6688, (1995). Typically, the anti-receptor peptide or apeptide analog is used either alone or conjugated to an immunogeniccarrier, as the immunogen for producing anti-receptor peptide monoclonalantibodies.

There are also a number of other well-known techniques for makingmonoclonal antibodies as binding molecules in this invention.Particularly useful are methods of making fully human antibodies. Onemethod is phage display technology which can be used to select a rangeof human antibodies binding specifically to the antigen using methods ofaffinity enrichment. Phage display has been thoroughly described in theliterature and the construction and screening of phage display librariesare well known in the art, see, e.g., Dente et al, Gene. 148(1):7-13(1994); Little et al, Biotechnol Adv. 12(3):539-55 (1994); Clackson etal., Nature 352:264-628 (1991); Huse et al., Science 246:1275-1281(1989).

Monocolonal antibodies derived by hybridoma technique from anotherspecies than human, such as mouse, can be humanized to avoid humananti-mouse antibodies when infused into humans. Among the more commonmethods of humanization of antibodies are complementarity-determiningregion grafting and resurfacing. These methods have been extensivelydescribed, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et al,Immunol Rev. 222:9-27 (2008); Almagro et al, Front Biosci. 1; 13:1619-33(2008); Lazar et al, Mol Immunol. 44(8):1986-98 (2007); Li et al, Proc.Natl. Acad. Sci. USA. 103(10):3557-62 (2006) each incorporated herein byreference. Fully human antibodies can also be prepared by immunizingtransgenic mice, rabbits, monkeys, or other mammals, carrying largeportions of the human immunoglobulin heavy and light chains, with animmunogen. Examples of such mice are: the Xenomouse. (Abgenix, Inc.),the HuMAb-Mouse (Medarex/BMS), the VelociMouse (Regeneron), see alsoU.S. Pat. Nos. 6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181,5,922,545, 5,661,016, 5,545,806, 5,436,149 and 5,569,825. In humantherapy, murine variable regions and human constant regions can also befused to construct called “chimeric antibodies” that are considerablyless immunogenic in man than murine mAbs (Kipriyanov et al, MolBiotechnol. 26:39-60 (2004); Houdebine, Curr Opin Biotechnol. 13:625-9(2002) each incorporated herein by reference). In addition,site-directed mutagenesis in the variable region of an antibody canresult in an antibody with higher affinity and specificity for itsantigen (Brannigan et al, Nat Rev Mol Cell Biol. 3:964-70, (2002));Adams et al, J Immunol Methods. 231:249-60 (1999)) and exchangingconstant regions of a mAb can improve its ability to mediate effectorfunctions of binding and cytotoxicity.

Antibodies immunospecific for a malignant cell antigen can also beobtained commercially or produced by any method known to one of skill inthe art such as, e.g., chemical synthesis or recombinant expressiontechniques. The nucleotide sequence encoding antibodies immunospecificfor a malignant cell antigen can be obtained commercially, e.g., fromthe GenBank database or a database like it, the literature publications,or by routine cloning and sequencing.

Apart from an antibody, a peptide or protein that bind/block/target orin some other way interact with the epitopes or corresponding receptorson a targeted cell can be used as a binding molecule. These peptides orproteins could be any random peptide or proteins that have an affinityfor the epitopes or corresponding receptors and they don't necessarilyhave to be of the immunoglobulin family. These peptides can be isolatedby similar techniques as for phage display antibodies (Szardenings, JRecept Signal Transduct Res. 2003; 23(4):307-49). The use of peptidesfrom such random peptide libraries can be similar to antibodies andantibody fragments. The binding molecules of peptides or proteins may beconjugated on or linked to a large molecules or materials, such as, butis not limited, an albumin, a polymer, a liposome, a nano particle, aslong as such attachment permits the peptide or protein to retain itsantigen binding specificity.

Examples of antibodies used for conjugation of antimitotic agents inthis prevention for treating cancer, autoimmune disease, and infectiousdisease include, but are not limited to, 3F8 (anti-GD2), Abagovomab(anti CA-125), Abciximab (anti CD41 (integrin alpha-IIb), Adalimumab(anti-TNF-α), Adecatumumab (anti-EpCAM, CD326), Afelimomab (anti-TNF-α);Afutuzumab (anti-CD20), Alacizumab pegol (anti-VEGFR2), ALD518(anti-IL-6), Alemtuzumab (Campath, MabCampath, anti-CD52), Altumomab(anti-CEA), Anatumomab (anti-TAG-72), Anrukinzumab (IMA-638,anti-IL-13), Apolizumab (anti-HLA-DR), Arcitumomab (anti-CEA),Aselizumab (anti-L-selectin (CD62L), Atlizumab (tocilizumab, Actemra,RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rhesus factor),Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, antiCD25 (αchain of IL-2 receptor), Bavituximab (anti-phosphatidylserine),Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B,anti-BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCL11(eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen),Bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti-fibrinII beta chain), Bivatuzumab (anti-CD44 v6), Blinatumomab (BiTE,anti-CD19), Brentuximab (cAC10, anti-CD30 TNFRSF8), Briakinumab(anti-IL-12, IL-23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242,anti-CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49(anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab pegol (Cimziaanti-TNF-α), Cetuximab (Erbitux, IMC-C225, anti-EGFR), Citatuzumabbogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4),Clivatuzumab (anti-MUC1), Conatumumab (anti-TRAIL-R₂), CR6261(anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab(Zenapax, anti-CD25 (α chain of IL-2 receptor)), Daratumumab (anti-CD38(cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab(anti-B-lymphoma cell), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab(anti-endotoxin), Edrecolomab (Panorex, MAb17-1A, anti-EpCAM),Efalizumab (Raptiva, anti-LFA-1 (CD11a), Efungumab (Mycograb,anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6),Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin),Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab(Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti-integrinα_(v)β₃), Exbivirumab (anti-hepatitis B surface antigen), Fanolesomab(NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor),Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratorysyncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1receptor), Fontolizumab (anti-IFN-γ), Foravirumab (anti-rabies virusglycoprotein), Fresolimumab (anti-TGF-β), Galiximab (anti-CD80),Gantenerumab (anti-beta amyloid), Gavilimomab (anti-CD147 (basigin)),Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9),Glembatumumab (CR011, anti-GPNMB), Golimumab (Simponi, anti-TNF-α),Gomiliximab (anti-CD23 (IgE receptor)), anti-HLA-DR antibody, Ibalizumab(anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125,anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab(Remicade, anti-TNF-α), Intetumumab (anti-CD51), Inolimomab (anti-CD25(a chain of IL-2 receptor)), Inotuzumab (anti-CD22), Ipilimumab(anti-CD152), Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4),Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalesomab(anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2),Lexatumumab (anti-TRAIL-R₂), Libivirumab (anti-hepatitis B surfaceantigen), Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab(anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R₁), Maslimomab(anti-T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria,anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74),Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside),Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti-respiratorysyncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab(anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrinα₄), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab(anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab,Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (CD11a)),Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R α), Omalizumab(Xolair, anti-IgE Fc region), Oportuzumab (anti-EpCAM), Oregovomab(OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab(anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis,anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF,anti-EGFR), Panobacumab (anti-Pseudomonas aeruginosa), Pascolizumab(anti-IL-4), Pemtumomab (Theragyn, anti-MUC1), Pertuzumab (Omnitarg,2C4, anti-HER2/neu), Pexelizumab (anti-C5), Pintumomab(anti-adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab(anti-vimentin), PRO 140 (anti-CCR5), Racotumomab (1E10,anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)),Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2),Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin,protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B),Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera,Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab(anti-IFN-α), Rovelizumab (LeukArrest, anti-CD11, CD18), Ruplizumab(Antova, anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab(anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab(anti-IFN-α), Siltuximab (anti-IL-6), Siplizumab (anti-CD2), (Smart)MI95 (anti-CD33), Solanezumab (anti-beta amyloid), Sonepcizumab(anti-sphingosine-1-phosphate), Sontuzumab (anti-episialin), Stamulumab(anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyteantigen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab(anti-integrin α_(IIb)β₃), Talizumab (anti-IgE), Tanezumab (anti-NGF),Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping factorA), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40),Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab,(anti-CTLA-4), Tigatuzumab (anti-TRAIL-R₂), TNX-650 (anti-IL-13),Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor),Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab(Herceptin, (anti-HER2/neu), Tremelimumab (anti-CTLA-4), Tucotuzumabcelmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus),Urtoxazumab (anti-Escherichia coli), Ustekinumab (Stelara, anti-IL-12,IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab, (anti-integrinα₄β₇), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1),Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3),Volociximab (anti-integrin α₅β₁), Votumumab (HumaSPECT, anti-tumorantigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab(HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147 (basigin)), Zolimomab(anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept(Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron RegulatoryProtein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst. formelanoma and solid tumors), J591 (anti-PSMA, Weill Cornell MedicalSchool for prostate cancers), 225.28S [anti-HMW-MAA (High molecularweight-melanoma-associated antigen), Sorin Radiofarmaci S.R.L. (Milan,Italy) for melanoma], COL-1 (anti-CEACAM3, CGM1, from Nat. Cancer Inst.USA for colorectal and gastric cancers), CYT-356 (Oncoltad®, forprostate cancers), HNK20 (OraVax Inc. for respiratory syncytial virus),ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, PeregrinePharm. for Cancers), MAK-195F [anti-TNF (tumor necrosis factor; TNFA,TNF-alpha; TNFSF2), from Abbott/Knoll for Sepsis toxic shock], MEDI-500[T10B9, anti-CD3, TRαβ (T cell receptor alpha/beta), complex, fromMedImmune Inc for Graft-versus-host disease], RING SCAN [anti-TAG 72(tumor associated glycoprotein 72), from Neoprobe Corp. for Breast,Colon and Rectal cancers], Avicidin (anti-EPCAM (epithelial celladhesion molecule), anti-TACSTD1 (Tumor-associated calcium signaltransducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS1/4 antigen;M4S; tumor antigen 17-1A; CD326, from NeoRx Corp. for Colon, Ovarian,Prostate cancers and NHL]; anti-Trop-2-humanized antibody hRS7,LymphoCide (Immunomedics, NJ), Smart ID 10 (Protein Design Labs),Oncolym (Techniclone Inc, CA), Allomune (BioTransplant, CA), anti-VEGF(Genentech, CA); CEAcide (Immunomedics, NJ), IMC-1C11 (ImClone Systems)and Cetuximab (ImClone).

Other antibodies as binding ligands include, but are not limited to, areantibodies against the following antigens: Aminopeptidase N (CD13),Annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian), CA15-3(carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y(carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas),CA242 (colorectal), placental alkaline phosphatase (carcinomas),prostate specific antigen (prostate), prostatic acid phosphatase(prostate), epidermal growth factor (carcinomas), CD2 (Hodgkin'sdisease, NHL lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma,lung, breast, gastric, ovarian cancers, autoimmune diseases, malignantascites), CD19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma),CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin'slymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiplemyeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51(Metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lungcancers, ovarian cancer, Merkel cell carcinoma, and the liquid tumor,multiple myeloma), CD66e (cancers), CD70 (metastatic renal cellcarcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80(lymphoma), CD98 (cancers), mucin (carcinomas), CD221 (solid tumors),CD227 (breast, ovarian cancers), CD262 (NSCLC and other cancers), CD309(ovarian cancers), CD326 (solid tumors), CEACAM3 (colorectal, gastriccancers), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast,colorectal and lung cancers), DLL4 (A-like-4), EGFR (Epidermal GrowthFactor Receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184,Heme-oncology, solid tumors), Endoglin (CD105, solid tumors), EPCAM(epithelial cell adhesion molecule, bladder, head, neck, colon, NHLprostate, and ovarian cancers), ERBB2 (Epidermal Growth Factor Receptor2; lung, breast, prostate cancers), FCGR1 (autoimmune diseases), FOLR(folate receptor, ovarian cancers), GD2 ganglioside (cancers), G-28 (acell surface antigen glyvolipid, melanoma), GD3 idiotype (cancers), Heatshock proteins (cancers), HER1 (lung, stomach cancers), HER2 (breast,lung and ovarian cancers), HLA-DR10 (NHL), HLA-DRB (NHL, B cellleukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-likegrowth factor 1 receptor, solid tumors, blood cancers), IL-2 receptor(interleukin 2 receptor, T-cell leukemia and lymphomas), IL-6R(interleukin 6 receptor, multiple myeloma, RA, Castleman's disease, IL6dependent tumors), Integrins (αvβ3, α5β1, α6β4, α11β3, α5β5, αvβ5, forvarious cancers), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3(carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor(carcinomas), p97 (melanoma), MS4A1 (membrane-spanning 4-domainssubfamily A member 1, Non-Hodgkin's B cell lymphoma, leukemia), MUC1 orMUC1-KLH (breast, ovarian, cervix, bronchus and gastrointestinalcancer), MUC16 (CA125) (Ovarian cancers), CEA (colorectal), gp100(melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (membrane-spanning4-domains subfamily A, small cell lung cancers, NHL), Nucleolin, Neuoncogene product (carcinomas), P21 (carcinomas), Paratope ofanti-(N-glycolylneuraminic acid, Breast, Melanoma cancers), PLAP-liketesticular alkaline phosphatase (ovarian, testicular cancers), PSMA(prostate tumors), PSA (prostate), ROBO4, TAG 72 (tumour associatedglycoprotein 72, AML, gastric, colorectal, ovarian cancers), T celltransmembrane protein (cancers), Tie (CD202b), TNFRSF10B (tumor necrosisfactor receptor superfamily member 10B, cancers), TNFRSF13B (tumornecrosis factor receptor superfamily member 13B, multiple myeloma, NHL,other cancers, RA and SLE), TPBG (trophoblast glycoprotein, Renal cellcarcinoma), TRAIL-R1 (Tumor necrosis apoprosis Inducing ligand Receptor1, lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma),VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumorassociated antigens recognized by antibodies have been reviewed (Gerber,et al, mAbs 1:3, 247-253 (2009); Novellino et al, Cancer ImmunolImmunother. 54(3), 187-207 (2005). Franke, et al, Cancer BiotherRadiopharm. 2000, 15, 459-76). Examples of these antigens thatantibodies against are: Many other Cluster of Differentiations (CD4,CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15,CD16, CDw17, CD18, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD31,CD32, CD34, CD35, CD36, CD37, CD41, CD42, CD43, CD44, CD45, CD46, CD47,CD48, CD49b, CD49c, CD53, CD54, CD55, CD58, CD59, CD61, CD62E, CD62L,CD62P, CD63, CD68, CD69, CD71, CD72, CD79, CD81, CD82, CD83, CD86, CD87,CD88, CD89, CD90, CD91, CD95, CD96, CD100, CD103, CD105, CD106, CD109,CD117, CD120, CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143,CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163, CD166, CD168,CD184, CDw186, CD195, CD202 (a, b), CD209, CD235a, CD271, CD303, CD304),Annexin A1, Nucleolin, Endoglin (CD105), ROBO4, Amino-peptidase N,Δ-like-4 (DLL4), VEGFR-2 (CD309), CXCR4 9CD184), Tie2, B7-H3, WT1, MUC1,LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53 nonmutant,NY-ESO-1, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant,Proteinase3 (PR1), bcr-abl, Tyrosinase, Survivin, hTERT, Sarcomatranslocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, ALK, Androgen receptor, Cyclin B1,Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA,MAGE A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML,NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Spermprotein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2,Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-P3, MAD-CT-2, Fos-related antigen 1.

In another specific embodiment, the antimitotic agent-binding moleculeconjugates of the invention are used in accordance with the compositionsand methods of the invention for the treatment of cancers. The cancersinclude, but are not limited, Adrenocortical Carcinoma, Anal Cancer,Bladder Cancer, Brain Tumor (Adult, Brain Stem Glioma, Childhood,Cerebellar Astrocytoma, Cerebral Astrocytoma, Ependymoma,Medulloblastoma, Supratentorial Primitive Neuroectodermal and PinealTumors, Visual Pathway and Hypothalamic Glioma), Breast Cancer,Carcinoid Tumor, Gastrointestinal, Carcinoma of Unknown Primary,Cervical Cancer, Colon Cancer, Endometrial Cancer, Esophageal Cancer,Extrahepatic Bile Duct Cancer, Ewings Family of Tumors (PNET),Extracranial Germ Cell Tumor, Eye Cancer, Intraocular Melanoma,Gallbladder Cancer, Gastric Cancer (Stomach), Germ Cell Tumor,Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer,Hypopharyngeal Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cellcancer), Laryngeal Cancer, Leukemia (Acute Lymphoblastic, Acute Myeloid,Chronic Lymphocytic, Chronic Myelogenous, Hairy Cell), Lip and OralCavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell,Lymphoma (AIDS-Related, Central Nervous System, Cutaneous T-Cell,Hodgkin's Disease, Non-Hodgkin's Disease, Malignant Mesothelioma,Melanoma, Merkel Cell Carcinoma, Metastatic Squamous Neck Cancer withOccult Primary, Multiple Myeloma, and Other Plasma Cell Neoplasms,Mycosis Fungoides, Myelodysplastic Syndrome, MyeloproliferativeDisorders, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer,Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer (Epithelial, GermCell Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine,Islet Cell Carcinoma), Paranasal Sinus and Nasal Cavity Cancer,Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer, PituitaryCancer, Plasma Cell Neoplasm, Prostate Cancer Rhabdomyosarcoma, RectalCancer, Renal Cell Cancer (kidney cancer), Renal Pelvis and Ureter(Transitional Cell), Salivary Gland Cancer, Sezary Syndrome, SkinCancer, Skin Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma,Melanoma), Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer,Testicular Cancer, Thymoma (Malignant), Thyroid Cancer, Urethral Cancer,Uterine Cancer (Sarcoma), Unusual Cancer of Childhood, Vaginal Cancer,Vulvar Cancer, Wilms' Tumor.

In another specific embodiment, the antimitotic agent-binding moleculeconjugates of the invention are used in accordance with the compositionsand methods of the invention for the treatment or prevention of anautoimmune disease. The autoimmune diseases include, but are notlimited, Achlorhydra Autoimmune Active Chronic Hepatitis, AcuteDisseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis,Addison's Disease, Agammaglobulinemia, Alopecia areata, AmyotrophicLateral Sclerosis, Ankylosing Spondylitis, Anti-GBM/TBM Nephritis,Antiphospholipid syndrome, Antisynthetase syndrome, Arthritis, Atopicallergy, Atopic Dermatitis, Autoimmune Aplastic Anemia, Autoimmunecardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis,Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome,Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmunepolyendocrine syndrome Types I, II, & III, Autoimmune progesteronedermatitis, Autoimmune thrombocytopenic purpura, Autoimmune uveitis,Balo disease/Balo concentric sclerosis, Bechets Syndrome, Berger'sdisease, Bickerstaffs encephalitis, Blau syndrome, Bullous Pemphigoid,Castleman's disease, Chagas disease, Chronic Fatigue Immune DysfunctionSyndrome, Chronic inflammatory demyelinating polyneuropathy, Chronicrecurrent multifocal ostomyelitis, Chronic lyme disease, Chronicobstructive pulmonary disease, Churg-Strauss syndrome, CicatricialPemphigoid, Coeliac Disease, Cogan syndrome, Cold agglutinin disease,Complement component 2 deficiency, Cranial arteritis, CREST syndrome,Crohns Disease (a type of idiopathic inflammatory bowel diseases),Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease,Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetesmellitus type 1, Diffuse cutaneous systemic sclerosis, Dressler'ssyndrome, Discoid lupus erythematosus, Eczema, Endometriosis,Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysisbullosa acquisita, Erythema nodosum, Essential mixed cryoglobulinemia,Evan's syndrome, Fibrodysplasia ossificans progressiva, Fibromyalgia,Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinalpemphigoid, Giant cell arteritis, Glomerulonephritis, Goodpasture'ssyndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto'sencephalitis, Hashimoto's thyroiditis, Haemolytic anaemia,Henoch-Schonlein purpura, Herpes gestationis, Hidradenitis suppurativa,Hughes syndrome (See Antiphospholipid syndrome), Hypogammaglobulinemia,Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonaryfibrosis, Idiopathic thrombocytopenic purpura (See Autoimmunethrombocytopenic purpura), IgA nephropathy (Also Berger's disease),Inclusion body myositis, Inflammatory demyelinating polyneuopathy,Interstitial cystitis, Irritable Bowel Syndrome, Juvenile idiopathicarthritis, Juvenile rheumatoid arthritis, Kawasaki's Disease,Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichenplanus, Lichen sclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease(Also Amyotrophic lateral sclerosis), Lupoid hepatitis, Lupuserythematosus, Majeed syndrome, Meniere's disease, Microscopicpolyangiitis, Miller-Fisher syndrome, Mixed Connective Tissue Disease,Morphea, Mucha-Habermann disease, Muckle-Wells syndrome, MultipleMyeloma, Multiple Sclerosis, Myasthenia gravis, Myositis, Narcolepsy,Neuromyelitis optica (Devic's Disease), Neuromyotonia, Occularcicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis,Palindromic rheumatism, PANDAS (Pediatric Autoimmune NeuropsychiatricDisorders Associated with Streptococcus), Paraneoplastic cerebellardegeneration, Paroxysmal nocturnal hemoglobinuria, Parry Rombergsyndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus,Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis,POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica,Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis,Progressive inflammatory neuropathy, Psoriasis, Psoriatic Arthritis,Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis,Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome,Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis,Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt syndrome,Schnitzler syndrome, Scleritis, Scleroderma, Sjögren's syndrome,Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiffperson syndrome, Subacute bacterial endocarditis, Susac's syndrome,Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu'sarteritis, Temporal arteritis (giant cell arteritis), Tolosa-Huntsyndrome, Transverse Myelitis, Ulcerative Colitis (a type of idiopathicinflammatory bowel diseases), Undifferentiated connective tissuedisease, Undifferentiated spondyloarthropathy, Vasculitis, Vitiligo,Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome

In another specific embodiment, a binding molecule used for theconjugate for the treatment or prevention of an autoimmune diseaseincludes, but are not limited to, anti-elastin antibody; Abys againstepithelial cells antibody; Anti-Basement Membrane Collagen Type IVProtein antibody; Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, AntiCardiolipin Antibody IgM, IgG; anti-celiac antibody; Anti PhospholipidAntibody IgK, IgG; Anti SM Antibody; Anti Mitochondrial Antibody;Thyroid Antibody; Microsomal Antibody, T-cells antibody; ThyroglobulinAntibody, Anti SCL-70; Anti-Jo; Anti-U.sub. RNP; Anti-La/SSB; Anti SSA;Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA;P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody,Anti-ganglioside antibody; Anti-Desmogein 3 antibody; Anti-p62 antibody;Anti-sp100 antibody; Anti-Mitochondrial (M2) antibody; Rheumatoid factorantibody; Anti-MCV antibody; Anti-topoisomerase antibody;Anti-neutrophil cytoplasmic (cANCA) antibody.

In certain preferred embodiments, the binding molecule for the conjugatein the present invention, can bind to both a receptor or a receptorcomplex expressed on an activated lymphocyte which is associated with anautoimmune disease. The receptor or receptor complex can comprise animmunoglobulin gene superfamily member (e.g. CD2, CD3, CD4, CD8, CD19,CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1, or ICOS), a TNF receptorsuperfamily member (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB,INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1,TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3), an integrin, a cytokinereceptor, a chemokine receptor, a major histocompatibility protein, alectin (C-type, S-type, or I-type), or a complement control protein.

In another specific embodiment, useful binding ligands that areimmunospecific for a viral or a microbial antigen are humanized or humanmonoclonal antibodies. As used herein, the term “viral antigen”includes, but is not limited to, any viral peptide, polypeptide protein(e.g. HIV gp120, HIV nef, RSV F glycoprotein, influenza virusneuramimidase, influenza virus hemagglutinin, HTLV tax, herpes simplexvirus glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surfaceantigen) that is capable of eliciting an immune response. As usedherein, the term “microbial antigen” includes, but is not limited to,any microbial peptide, polypeptide, protein, saccharide, polysaccharide,or lipid molecule (e.g., a bacterial, fungi, pathogenic protozoa, oryeast polypeptide including, e.g., LPS and capsular polysaccharide 5/8)that is capable of eliciting an immune response. Examples of antibodiesavailable 1 for the viral or microbial infection include, but are notlimited to, Palivizumab which is a humanized anti-respiratory syncytialvirus monoclonal antibody for the treatment of RSV infection; PRO542which is a CD4 fusion antibody for the treatment of HIV infection;Ostavir which is a human antibody for the treatment of hepatitis Bvirus; PROTVIR which is a humanized IgG.sub.1 antibody for the treatmentof cytomegalovirus; and anti-LPS antibodies.

The binding molecules-antimitotic agent conjugates of this invention canbe used in the treatment of infectious diseases. These infectiousdiseases include, but are not limited to, Acinetobacter infections,Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS(Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax,Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever,Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacilluscereus infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroidesinfection, Balantidiasis, Baylisascaris infection, BK virus infection,Black piedra, Blastocystis hominis infection, Blastomycosis, Bolivianhemorrhagic fever, Borrelia infection, Botulism (and Infant botulism),Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Buruliulcer, Calicivirus infection (Norovirus and Sapovirus),Campylobacteriosis, Candidiasis (Moniliasis; Thrush), Cat-scratchdisease, Cellulitis, Chagas Disease (American trypanosomiasis),Chancroid, Chickenpox, Chlamydia, Chlamydophila pneumoniae infection,Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficileinfection, Coccidioidomycosis, Colorado tick fever, Common cold (Acuteviral rhinopharyngitis; Acute coryza), Creutzfeldt-Jakob disease,Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis,Cutaneous larva migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirusinfection, Dengue fever, Dientamoebiasis, Diphtheria,Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever,Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection),Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythemainfectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis,Fasciolosis, Fatal familial insomnia, Filariasis, Food poisoning byClostridium perfringens, Free-living amebic infection, Fusobacteriuminfection, Gas gangrene (Clostridial myonecrosis), Geotrichosis,Gerstmann-Sträussler-Scheinker syndrome, Giardiasis, Glanders,Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group Astreptococcal infection, Group B streptococcal infection, Haemophilusinfluenzae infection, Hand, foot and mouth disease (HFMD), HantavirusPulmonary Syndrome, Helicobacter pylori infection, Hemolytic-uremicsyndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, HepatitisB, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex,Histoplasmosis, Hookworm infection, Human bocavirus infection, Humanewingii ehrlichiosis, Human granulocytic anaplasmosis, Humanmetapneumovirus infection, Human monocytic ehrlichiosis, Humanpapillomavirus infection, Human parainfluenza virus infection,Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono),Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingaeinfection, Kuru, Lassa fever, Legionellosis (Legionnaires' disease),Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis,Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis(Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburghemorrhagic fever, Measles, Melioidosis (Whitmore's disease),Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis,Molluscum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasmapneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmianeonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD),Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis(South American blastomycosis), Paragonimiasis, Pasteurellosis,Pediculosis capitis (Head lice), Pediculosis corporis (Body lice),Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease,Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystispneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primaryamoebic meningoencephalitis, Progressive multifocal leukoencephalopathy,Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytialvirus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsialinfection, Rickettsialpox, Rift Valley fever, Rocky mountain spottedfever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe AcuteRespiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis(Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola),Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection,Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae(Barber's itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis(Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringwormof the Hand), Tinea nigra, Tinea pedis (Athlete's foot), Tinea unguium(Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis(Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans),Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworminfection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection,Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Viralpneumonia, West Nile Fever, White piedra (Tinea blanca), Yersiniapseudotuberculosis infection, Yersiniosis, Yellow fever, Zygomycosis.

The binding molecules, proffered antibodies described in this patentthat are against pathogenic strains include, but are not limit,Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriaeand Propionibacterium propionicus, Trypanosoma brucei, HIV (Humanimmunodeficiency virus), Entamoeba histolytica, Anaplasma genus,Bacillus anthracis, Arcanobacterium haemolyticum, Junin virus, Ascarislumbricoides, Aspergillus genus, Astroviridae family, Babesia genus,Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli,Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis,Blastomyces dermatitides, Machupo virus, Borrelia genus, Clostridiumbotulinum, Sabia, Brucella genus, usually Burkholderia cepacia and otherBurkholderia species, Mycobacterium ulcerans, Caliciviridae family,Campylobacter genus, usually Candida albicans and other Candida species,Bartonella henselae, Group A Streptococcus and Staphylococcus,Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV),Chlamydia trachomatis, Chlamydophila pneumoniae, Vibrio cholerae,Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile,Coccidioides immitis and Coccidioides posadasii, Colorado tick fevervirus, rhinoviruses, coronaviruses, CJD prion, Crimean-Congo hemorrhagicfever virus, Cryptococcus neoformans, Cryptosporidium genus, Ancylostomabraziliense; multiple parasites, Cyclospora cayetanensis, Taenia solium,Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 andDEN-4)—Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae,Diphyllobothrium, Dracunculus medinensis, Ebolavirus, Echinococcusgenus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus,Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Humanherpesvirus 6 and Human herpesvirus 7, Fasciolopsis buski, Fasciolahepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily,Clostridium perfringens, Fusobacterium genus, Clostridium perfringens;other Clostridium species, Geotrichum candidum, GSS prion, Giardiaintestinalis, Burkholderia mallei, Gnathostoma spinigerum andGnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis,Streptococcus pyogenes, Streptococcus agalactiae, Haemophilusinfluenzae, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71,Sin Nombre virus, Helicobacter pylori, Escherichia coli O157:H7,Bunyaviridae family, Hepatitis A Virus, Hepatitis B Virus, Hepatitis CVirus, Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1,Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenaleand Necator americanus, Hemophilus influenzae, Human bocavirus,Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus,Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenzaviruses, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Virus,Orthomyxoviridae family, Isospora belli, Kingella kingae, Klebsiellapneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion,Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmaniagenus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospiragenus, Listeria monocytogenes, Borrelia burgdorferi and other Borreliaspecies, Wuchereria bancrofti and Brugia malayi, Lymphocyticchoriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measlesvirus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous speciesof bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterousfly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion,Nocardia asteroides and other Nocardia species, Onchocerca volvulus,Paracoccidioides brasiliensis, Paragonimus westermani and otherParagonimus species, Pasteurella genus, Pediculus humanus capitis,Pediculus humanus corporis, Phthirus pubis, Bordetella pertussis,Yersinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii,Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophilapsittaci, Coxiella burnetii, Rabies virus, Streptobacillus moniliformisand Spirillum minus, Respiratory syncytial virus, Rhinosporidiumseeberi, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valleyfever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonellagenus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigellagenus, Varicella zoster virus, Variola major or Variola minor,Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus,Staphylococcus aureus, Streptococcus pyogenes, Strongyloidesstercoralis, Treponema pallidum, Taenia genus, Clostridium tetani,Trichophyton genus, Trichophyton tonsurans, Trichophyton genus,Epidermophyton floccosum, Trichophyton rubrum, and Trichophytonmentagrophytes, Trichophyton rubrum, Hortaea werneckii, Trichophytongenus, Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasmagondii, Trichinella spiralis, Trichomonas vaginalis, Trichuristrichiura, Mycobacterium tuberculosis, Francisella tularensis,Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibriocolerae, Guanarito virus, West Nile virus, Trichosporon beigelii,Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fevervirus, Mucorales order (Mucormycosis) and Entomophthorales order(Entomophthoramycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio)fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis,Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonellatyphimurium, Treponema pertenue, Treponema carateneum, Borreliavincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,Pneumocystis carinii, Brucella abortus, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus,Candida albicans, Histoplasma capsulatum); protozoa (Entomoebahistolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosomagambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmaniatropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodiumvivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths(Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium,and hookworms).

Other antibodies as a binding ligand in this invention for treatment ofviral disease include, but are not limited to, antibodies againstantigens of pathogenic viruses, including as examples and not bylimitation: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae,Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae,influenza viruses, parainfluenza viruses, mumps, measles, respiratorysyncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae,Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae,Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervicalcancer, Anal cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi'ssarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt'slymphoma, Primary central nervous system lymphoma), MCPyV (Merkel cellcancer), SV40 (Simian virus 40), HCV (Hepatocellular carcinoma), HTLV-I(Adult T-cell leukemia/lymphoma)], Immune disorders caused virus: [suchas Human Immunodeficiency Virus (AIDS)]; Central nervous system virus:[such as, JCV (Progressive multifocal leukoencephalopathy), MeV(Subacute sclerosing panencephalitis), LCV (Lymphocyticchoriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (probable)(Encephalitis lethargica), RV (Rabies), Chandipura virus, Herpesviralmeningitis, Ramsay Hunt syndrome type II; Poliovirus (Poliomyelitis,Post-polio syndrome), HTLV-I (Tropical spastic paraparesis)];Cytomegalovirus (Cytomegalovirus retinitis, HSV (Herpetic keratitis));Cardiovascular virus [such as CBV (Pericarditis, Myocarditis)];Respiratory system/acute viral nasopharyngitis/viral pneumonia:[Epstein-Barr virus (EBV infection/Infectious mononucleosis),Cytomegalovirus; SARS coronavirus (Severe acute respiratory syndrome)Orthomyxoviridae: Influenzavirus A/B/C (Influenza/Avian influenza),Paramyxovirus: Human parainfluenza viruses (Parainfluenza), RSV (Humanrespiratory syncytial virus), hMPV]; Digestive system virus [MuV(Mumps), Cytomegalovirus (Cytomegalovirus esophagitis); Adenovirus(Adenovirus infection); Rotavirus, Norovirus, Astrovirus, Coronavirus;HBV (Hepatitis B virus), CBV, HAV (Hepatitis A virus), HCV (Hepatitis Cvirus), HDV (Hepatitis D virus), HEV (Hepatitis E virus), HGV (HepatitisG virus)]; Urogenital virus [such as, BK virus, MuV (Mumps)].

According to a further object, the present invention also concernspharmaceutical compositions comprising the conjugate of the inventiontogether with a pharmaceutically acceptable carrier for treatment ofcancer and autoimmune disorders. The method for treatment of cancer andautoimmune disorders can be practiced in vitro, in vivo, or ex vivo.Examples of in vitro uses include treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.Examples of ex vivo uses include treatments of hematopoietic stem cells(HSC) prior to the performance of the transplantation (HSCT) into thesame patient in order to kill diseased or malignant cells. For instance,clinical ex vivo treatment to remove tumor cells or lymphoid cells frombone marrow prior to autologous transplantation in cancer treatment orin treatment of autoimmune disease, or to remove T cells and otherlymphoid cells from allogeneic bone marrow or tissue prior to transplantin order to prevent graft-versus-host disease, can be carried out asfollows. Bone marrow is harvested from the patient or other individualand then incubated in medium containing serum to which is added theconjugate of the invention, concentrations range from about 1 pM to 0.1mM, for about 15 minutes to about 48 hours at about 37° C. The exactconditions of concentration and time of incubation (=dose) are readilydetermined by the skilled clinicians. After incubation the bone marrowcells are washed with medium containing serum and returned to thepatient by i.v. infusion according to known methods. In circumstanceswhere the patient receives other treatment such as a course of ablativechemotherapy or total-body irradiation between the time of harvest ofthe marrow and reinfusion of the treated cells, the treated marrow cellsare stored frozen in liquid nitrogen using standard medical equipment.

Formulation and Application

The conjugates of the patent application are formulated to liquid, orsuitable to be lyophilized and subsequently be reconstituted to a liquidformulation. The contemplated excipients, which may be utilized in theaqueous pharmaceutical compositions of the patent application include,for example, flavoring agents, antimicrobial agents, sweeteners,antioxidants, antistatic agents, lipids such as phospholipids or fattyacids, steroids such as cholesterol, protein excipients such as serumalbumin (human serum albumin), recombinant human albumin, gelatin,casein, salt-forming counterions such sodium and the like. These andadditional known pharmaceutical excipients and/or additives suitable foruse in the formulations of the invention are known in the art, e.g., aslisted in “The Handbook of Pharmaceutical Excipients, 4^(th) edition,Rowe et al., Eds., American Pharmaceuticals Association (2003); andRemington: the Science and Practice of Pharmacy, 21^(th) edition,Gennaro, Ed., Lippincott Williams & Wilkins (2005).

A pharmaceutical container or vessel is used to hold the pharmaceuticalformulation of any of conjugates of the patent application. The vesselis a vial, bottle, pre-filled syringe, pre-filled or auto-injectorsyringe. The liquid formula can be freeze-dried or drum-dryed to a formof cake or powder in a borosilicate vial or soda lime glass vial. Thesolid powder can also be prepared by efficient spray drying, and thenpacked to a vial or a pharmaceutical container for storage anddistribution.

In a further embodiment, the invention provides a method for preparing aformulation comprising the steps of: (a) lyophilizing the formulationcomprising the conjugates, excipients, and a buffer system; and (b)reconstituting the lyophilized mixture of step (a) in a reconstitutionmedium such that the reconstituted formulation is stable. Theformulation of step (a) may further comprise a stabilizer and one ormore excipients selected from a group comprising bulking agent, salt,surfactant and preservative as hereinabove described. As reconstitutionmedia, several diluted organic acids or water, i.e. sterile water,bacteriostatic water for injection (BWFI) or may be used. Thereconstitution medium may be selected from water, i.e. sterile water,bacteriostatic water for injection (BWFI) or the group consisting ofacetic acid, propionic acid, succinic acid, sodium chloride, magnesiumchloride, acidic solution of sodium chloride, acidic solution ofmagnesium chloride and acidic solution of arginine, in an amount fromabout 10 to about 250 mM.

A liquid pharmaceutical formulation of the conjugates of the patentapplication should exhibit a variety of pre-defined characteristics. Oneof the major concerns in liquid drug products is stability, asproteins/antibodies tend to form soluble and insoluble aggregates duringmanufacturing and storage. In addition, various chemical reactions canoccur in solution (deamidation, oxidation, clipping, isomerization etc.)leading to an increase in degradation product levels and/or loss ofbioactivity. Preferably, a conjugate in either liquid or loyphilizateformulation should exhibit a shelf life of more than 6 months at 25° C.More preferred a conjugate in either liquid or loyphilizate formulationshould exhibit a shelf life of more than 12 months at 25° C. Mostpreferred liquid formulation should exhibit a shelf life of about 24 to36 months at 2-8° C. and the loyphilizate formulation should exhibit ashelf life of about preferably up to 60 months at 2-8° C. Both liquidand loyphilizate formulations should exhibit a shelf life for at leasttwo years at −20° C., or −70° C.

In certain embodiments, the formulation is stable following freezing (e.g., −20° C., or −70° C.) and thawing of the formulation, for examplefollowing 1, 2 or 3 cycles of freezing and thawing. Stability can beevaluated qualitatively and/or quantitatively in a variety of differentways, including evaluation of drug/antibody (protein) ratio andaggregate formation (for example using UV, size exclusionchromatography, by measuring turbidity, and/or by visual inspection); byassessing charge heterogeneity using cation exchange chromatography,image capillary isoelectric focusing (icIEF) or capillary zoneelectrophoresis; amino-terminal or carboxy-terminal sequence analysis;mass spectrometric analysis, or matrix-assisted laser desorptionionization/time-of-flight mass spectrometry (MALDI/TOF MS), orHPLC-MS/MS; SDS-PAGE analysis to compare reduced and intact antibody;peptide map (for example tryptic or LYS-C) analysis; evaluatingbiological activity or antigen binding function of the antibody; etc.Instability may involve any one or more of: aggregation, deamidation(e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization(e.g. Asp isomerization), clipping/hydrolysis/fragmentation (e.g. hingeregion fragmentation), succinimide formation, unpaired cysteine(s),N-terminal extension, C-terminal processing, glycosylation differences,etc.

A stable conjugate should also “retains its biological activity” in apharmaceutical formulation, if the biological activity of the conjugateat a given time, e. g. 12 month, within about 20%, preferably about 10%(within the errors of the assay) of the biological activity exhibited atthe time the pharmaceutical formulation was prepared as determined in anantigen binding assay, and/or in vitro, cytotoxic assay, for example.

For clinical in vivo use, the conjugate via the linkers of the inventionwill be supplied as solutions or as a lyophilized solid that can beredissolved in sterile water for injection. Examples of suitableprotocols of conjugate administration are as follows. Conjugates aregiven daily, weekly, biweekly, triweekly, once every four weeks ormonthly for 8˜54 weeks as an i.v. bolus. Bolus doses are given in 50 to1000 ml of normal saline to which human serum albumin (e.g. 0.5 to 1 mLof a concentrated solution of human serum albumin, 100 mg/mL) canoptionally be added. Dosages will be about 50 μg to 20 mg/kg of bodyweight per week, i.v. (range of 10 μg to 200 mg/kg per injection). 4˜54weeks after treatment, the patient may receive a second course oftreatment. Specific clinical protocols with regard to route ofadministration, excipients, diluents, dosages, times, etc., can bedetermined by the skilled clinicians.

Examples of medical conditions that can be treated according to the invivo or ex vivo methods of killing selected cell populations includemalignancy of any types of cancer, autoimmune diseases, graftrejections, and infections (viral, bacterial or parasite).

The amount of a conjugate which is required to achieve the desiredbiological effect, will vary depending upon a number of factors,including the chemical characteristics, the potency, and thebioavailability of the conjugates, the type of disease, the species towhich the patient belongs, the diseased state of the patient, the routeof administration, all factors which dictate the required dose amounts,delivery and regimen to be administered.

In general terms, the conjugates via the linkers of this invention maybe provided in an aqueous physiological buffer solution containing 0.1to 10% w/v conjugates for parenteral administration. Typical dose rangesare from 1 μg/kg to 0.1 g/kg of body weight daily; weekly, biweekly,triweekly, or monthly, a preferred dose range is from 0.01 mg/kg to 20mg/kg of body weight weekly, biweekly, triweekly, or monthly, anequivalent dose in a human. The preferred dosage of drug to beadministered is likely to depend on such variables as the type andextent of progression of the disease or disorder, the overall healthstatus of the particular patient, the relative biological efficacy ofthe compound selected, the formulation of the compound, the route ofadministration (intravenous, intramuscular, or other), thepharmacokinetic properties of the conjugates by the chosen deliveryroute, and the speed (bolus or continuous infusion) and schedule ofadministrations (number of repetitions in a given period of time).

The conjugates of the present invention are also capable of beingadministered in unit dose forms, wherein the term “unit dose” means asingle dose which is capable of being administered to a patient, andwhich can be readily handled and packaged, remaining as a physically andchemically stable unit dose comprising either the active conjugateitself, or as a pharmaceutically acceptable composition, as describedhereinafter. As such, typical total daily/weekly/biweekly/monthly doseranges are from 0.01 to 100 mg/kg of body weight. By way of generalguidance, unit doses for humans range from 1 mg to 3000 mg per day, orper week, per two weeks (biweekly), triweekly, or per month. Preferablythe unit dose range is from 1 to 500 mg administered one to four times amonth and even more preferably from 1 mg to 100 mg, once a week, or oncebiweekly, or once triweekly. Conjugates provided herein can beformulated into pharmaceutical compositions by admixture with one ormore pharmaceutically acceptable excipients. Such unit dose compositionsmay be prepared for use by oral administration, particularly in the formof tablets, simple capsules or soft gel capsules; or intranasally,particularly in the form of powders, nasal drops, or aerosols; ordermally, for example, topically in ointments, creams, lotions, gels orsprays, or via trans-dermal patches. The compositions may convenientlybe administered in unit dosage form and may be prepared by any of themethods well known in the pharmaceutical art, for example, as describedin Remington: The Science and Practice of Pharmacy, 21^(th) ed.;Lippincott Williams & Wilkins: Philadelphia, PA, 2005.

Preferred formulations include pharmaceutical compositions in which acompound of the present invention is formulated for oral or parenteraladministration. For oral administration, tablets, pills, powders,capsules, troches and the like can contain one or more of any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, or gum tragacanth; a diluent such asstarch or lactose; a disintegrant such as starch and cellulosederivatives; a lubricant such as magnesium stearate; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, or methylsalicylate. Capsules can be in the form of a hard capsule or softcapsule, which are generally made from gelatin blends optionally blendedwith plasticizers, as well as a starch capsule. In addition, dosage unitforms can contain various other materials that modify the physical formof the dosage unit, for example, coatings of sugar, shellac, or entericagents. Other oral dosage forms syrup or elixir may contain sweeteningagents, preservatives, dyes, colorings, and flavorings. In addition, theactive compounds may be incorporated into fast dissolve,modified-release or sustained-release preparations and formulations, andwherein such sustained-release formulations are preferably bi-modal.Preferred tablets contain lactose, cornstarch, magnesium silicate,croscarmellose sodium, povidone, magnesium stearate, or talc in anycombination.

Liquid preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. The liquidcompositions may also include binders, buffers, preservatives, chelatingagents, sweetening, flavoring and coloring agents, and the like.Non-aqueous solvents include alcohols, propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and organic esters such asethyl oleate. Aqueous carriers include mixtures of alcohols and water,buffered media, and saline. In particular, biocompatible, biodegradablelactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be useful excipients tocontrol the release of the active compounds. Intravenous vehicles caninclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Other potentiallyuseful parenteral delivery systems for these active compounds includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes.

Alternative modes of administration include formulations for inhalation,which include such means as dry powder, aerosol, or drops. They may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or oily solutions foradministration in the form of nasal drops, or as a gel to be appliedintranasally. Formulations for buccal administration include, forexample, lozenges or pastilles and may also include a flavored base,such as sucrose or acacia, and other excipients such as glycocholate.Formulations suitable for rectal administration are preferably presentedas unit-dose suppositories, with a solid based carrier, such as cocoabutter, and may include a salicylate. Formulations for topicalapplication to the skin preferably take the form of an ointment, cream,lotion, paste, gel, spray, aerosol, or oil. Carriers which can be usedinclude petroleum jelly, lanolin, polyethylene glycols, alcohols, ortheir combinations. Formulations suitable for transdermal administrationcan be presented as discrete patches and can be lipophilic emulsions orbuffered, aqueous solutions, dissolved and/or dispersed in a polymer oran adhesive.

In a specific embodiment, a conjugate of the invention is administeredconcurrently with the other known or will be known therapeutic agentssuch as the chemotherapeutic agent, the radiation therapy, immunotherapyagents, autoimmune disorder agents, anti-infectious agents or the otherantibody-drug conjugates, resulting in a synergistic effect. In anotherspecific embodiment, the synergistic drugs or radiation therapy areadministered prior or subsequent to administration of a conjugate, inone aspect at least an hour, 12 hours, a day, a week, biweeks, triweeks,a month, in further aspects several months, prior or subsequent toadministration of a conjugate of the invention.

In other embodiments, the synergistic drugs include, but not limited to:

1). Chemotherapeutic agents: a). Alkylating agents: such as Nitrogenmustards: chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, mannomustine, mitobronitol, melphalan, mitolactol,pipobroman, novembichin, phenesterine, prednimustine, thiotepa,trofosfamide, uracil mustard; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); Duocarmycin (includingthe synthetic analogues, KW-2189 and CBI-TMI); Benzodiazepine dimers(e.g., dimmers of pyrrolobenzodiazepine (PBD) or tomaymycin,indolinobenzodiazepines, imidazobenzothiadiazepines, oroxazolidino-benzodiazepines); Nitrosoureas: (carmustine, lomustine,chlorozotocin, fotemustine, nimustine, ranimustine); Alkylsulphonates:(busulfan, treosulfan, improsulfan and piposulfan); Triazenes:(dacarbazine); Platinum containing compounds: (carboplatin, cisplatin,oxaliplatin); aziridines, such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemel-amine, trietylenephosphoramide,triethylenethio-phosphaoramide and trimethylolomel-amine]; b). PlantAlkaloids: such as Vinca alkaloids: (vincristine, vinblastine,vindesine, vinorelbine, navelbin); Taxoids: (paclitaxel, docetaxol) andtheir analogs, Maytansinoids (DM1, DM2, DM3, DM4, maytansine andansamitocins) and their analogs, cryptophycins (particularlycryptophycin 1 and cryptophycin 8); epothilones, eleutherobin,discodermo-lide, bryostatins, dolostatins, auristatins, amatoxins,cephalostatins; pancratistatin; a sarcodictyin; spongistatin; c). DNATopoisomerase Inhibitors: such as [Epipodophyllins:(9-aminocamptothecin, camptothecin, crisnatol, daunomycin, etoposide,etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoicacids (retinols), teniposide, topotecan, 9-nitrocamptothecin (RFS2000)); mitomycins: (mitomycin C)]; d). Anti-metabolites: such as{[Anti-folate: DHFR inhibitors: (methotrexate, trimetrexate, denopterin,pteropterin, aminopterin (4-aminopteroic acid) or the other folic acidanalogues); IMP dehydrogenase Inhibitors: (mycophenolic acid,tiazofurin, ribavirin, EICAR); Ribonucleotide reductase Inhibitors:(hydroxyurea, deferoxamine)]; [Pyrimidine analogs: Uracil analogs:(ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil,floxuridine, ratitrexed (Tomudex)); Cytosine analogs: (cytarabine,cytosine arabinoside, fludarabine); Purine analogs: (azathioprine,fludarabine, mercaptopurine, thiamiprine, thioguanine)]; folic acidreplenisher, such as frolinic acid}; e). Hormonal therapies: such as{Receptor antagonists: [Anti-estrogen: (megestrol, raloxifene,tamoxifen); LHRH agonists: (goscrclin, leuprolide acetate);Anti-androgens: (bicalutamide, flutamide, calusterone, dromostanolonepropionate, epitiostanol, goserelin, leuprolide, mepitiostane,nilutamide, testolactone, trilostane and other androgens inhibitors)];Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060,cholecalciferol, ergocalciferol); Photodynamic therapies: (verteporfin,phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A);Cytokines: (Interferon-alpha, Interferon-gamma, tumor necrosis factor(TNFs), human proteins containing a TNF domain)]}; f). Kinaseinhibitors, such as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib,pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib,lapatinib, axitinib, pazopanib. vandetanib, E7080 (anti-VEGFR2),mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib(SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387,axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab,Ranibizumab, Panitumumab, ispinesib; g). A poly (ADP-ribose) polymerase(PARP) inhibitors, such as olaparib, niraparib, iniparib, talazoparib,veliparib, veliparib, CEP 9722 (Cephalon's), E7016 (Eisai's), BGB-290(BeiGene's), 3-aminobenzamide.

h). antibiotics, such as the enediyne antibiotics (e.g. calicheamicins,especially calicheamicin γ1, δ1, α1 and β1, see, e.g., J. Med. Chem., 39(11), 2103-2117 (1996), Angew Chem Intl. Ed. Engl. 33:183-186 (1994);dynemicin, including dynemicin A and deoxydynemicin; esperamicin,kedarcidin, C-1027, maduropeptin, as well as neocarzinostatinchromophore and related chromoprotein enediyne antiobioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin;chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin,nitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin,potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; i). Others:such as Polyketides (acetogenins), especially bullatacin andbullatacinone; gemcitabine, epoxomicins (e. g. carfilzomib), bortezomib,thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032,STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Yervoy,Isoprenylation inhibitors (such as Lovastatin), Dopaminergic neurotoxins(such as 1-methyl-4-phenylpyridinium ion), Cell cycle inhibitors (suchas staurosporine), Actinomycins (such as Actinomycin D, dactinomycin),Bleomycins (such as bleomycin A2, bleomycin B2, peplomycin),Anthracyclines (such as daunorubicin, doxorubicin (adriamycin),idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDRinhibitors (such as verapamil), Ca²⁺ ATPase inhibitors (such asthapsigargin), Histone deacetylase inhibitors (Vorinostat, Romidepsin,Panobinostat, Valproic acid, Mocetinostat (MGCD0103), Belinostat,PCI-24781, Entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101,sulforaphane, Trichostatin A); Thapsigargin, Celecoxib, glitazones,epigallocatechin gallate, Disulfiram, Salinosporamide A.; Anti-adrenals,such as aminoglutethimide, mitotane, trilostane; aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside,bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; eflornithine (DFMO), elfomithine; elliptinium acetate,etoglucid; gallium nitrate; gacytosine, hydroxyurea; ibandronate,lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine;pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verrucarin A, roridin A and anguidine); urethane, siRNA, antisensedrugs, and a nucleolytic enzyme.

2). An anti-autoimmune disease agent includes, but is not limited to,cyclosporine, cyclosporine A, aminocaproic acid, azathioprine,bromocriptine, chlorambucil, chloroquine, cyclophosphamide,corticosteroids (e.g. amcinonide, betamethasone, budesonide,hydrocortisone, flunisolide, fluticasone propionate, fluocortolonedanazol, dexamethasone, Triamcinolone acetonide, beclometasonedipropionate), DHEA, enanercept, hydroxychloroquine, infliximab,meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus,tacrolimus.

3). An anti-infectious disease agent includes, but is not limited to,a). Aminoglycosides: amikacin, astromicin, gentamicin (netilmicin,sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin,bekanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin,ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin,verdamicin; b). Amphenicols:azidamfenicol, chloramphenicol, florfenicol,thiamphenicol; c). Ansamycins: geldanamycin, herbimycin; d).Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin,meropenem, panipenem; e). Cephems: carbacephem (loracarbef),cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine,cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole,cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin,cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin,cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir,cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin,cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome,cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime,ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin,cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f).Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin),teicoplanin (dalbavancin), ramoplanin; g). Glycylcyclines: e. g.tigecycline; g). β-Lactamase inhibitors: penam (sulbactam, tazobactam),clavam (clavulanic acid); i). Lincosamides: clindamycin, lincomycin; j).Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA);k). Macrolides: azithromycin, cethromycin, clarithromycin,dirithromycin, erythromycin, flurithromycin, josamycin, ketolide(telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin,rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin,roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506),troleandomycin, telithromycin; l). Monobactams: aztreonam, tigemonam;m). Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampicillin(pivampicillin, hetacillin, bacampicillin, metampicillin,talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathinebenzylpenicillin, benzathine phenoxymethyl-penicillin, clometocillin,procaine benzylpenicillin, carbenicillin (carindacillin), cloxacillin,dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam),mezlocillin, meticillin, nafcillin, oxacillin, penamecillin, penicillin,pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin,sulbenicillin, temocillin, ticarcillin; o). Polypeptides: bacitracin,colistin, polymyxin B; p). Quinolones: alatrofloxacin, balofloxacin,ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin,enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin,grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin,marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin,ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin,sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q).Streptogramins: pristinamycin, quinupristin/dalfopristin); r).Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole,sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim,trimethoprim-sulfamethoxazole (co-trimoxazole); s). Steroidantibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline,chlortetracycline, clomocycline, demeclocycline, lymecycline,meclocycline, metacycline, minocycline, oxytetracycline,penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g.tigecycline); u). Other types of antibiotics: annonacin, arsphenamine,bactoprenol inhibitors (Bacitracin), DADAL/AR inhibitors (cycloserine),dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol,etoposide, faropenem, fusidic acid, furazolidone, isoniazid,laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesisinhibitors (e. g. fosfomycin), nitrofurantoin, paclitaxel,platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin(rifampin), tazobactam tinidazole, uvaricin;

4). Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc, maraviroc,vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integraseinhibitors: raltegravir, elvitegravir, globoidnan A; c). Maturationinhibitors: bevirimat, vivecon; d). Neuraminidase inhibitors:oseltamivir, zanamivir, peramivir; e). Nucleosides &nucleotides:abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine,cidofovir, clevudine, dexelvucitabine, didanosine (ddI), elvucitabine,emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU),3′-fluoro-substituted 2′,3′-dideoxynucleoside analogues (e.g.3′-fluoro-2′,3′-dideoxythymidine (FLT) and3′-fluoro-2′,3′-dideoxyguanosine (FLG), fomivirsen, ganciclovir,idoxuridine, lamivudine (3TC), 1-nucleosides (e.g. β-1-thymidine andβ-1-2′-deoxycytidine), penciclovir, racivir, ribavirin, stampidine,stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir,trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine(AZT); f). Non-nucleosides: amantadine, ateviridine, capravirine,diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol,emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod,interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205,peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod(R-848), tromantadine; g). Protease inhibitors: amprenavir, atazanavir,boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir,pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h).Other types of anti-virus drugs: abzyme, arbidol, calanolide a,ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate(EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea,KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin,seliciclib.

5). The radioisotopes for radiotherapy. Examples of radioisotopes(radionuclides) are ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc,¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, or ²¹³Bi.Radioisotope labeled antibodies are useful in receptor targeted imagingexperiments or can be for targeted treatment such as with theantibody-radioisotope conjugates (Wu et al (2005) Nature Biotechnology23(9): 1137-46). The cell binding molecules, e.g. an antibody can belabeled with ligand reagents that bind, chelate or otherwise complex aradioisotope metal, using the techniques described in Current Protocolsin Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience,New York, Pubs. (1991). Chelating ligands which may complex a metal ioninclude DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex.USA).

6). Another cell-binding molecule-drug conjugate as a synergy therapy.The preferred synergic conjugate can be a conjugate having a cytotoxicagent of a tubulysin analog, maytansinoid analog, taxanoid (taxane)analog, CC-1065 analog, daunorubicin and doxorubicin compound, amatoxinanalog, benzodiazepine dimer (e.g., dimers of pyrrolobenzodiazepine(PBD), tomaymycin, anthramycin, indolinobenzodiazepines,imidazobenzothiadiazepines, or oxazolidinobenzodiazepines),calicheamicins and the enediyne antibiotic compound, actinomycin,azaserine, bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins,auristatins (e.g. monomethyl auristatin E, MMAE, MMAF, auristatin PYE,auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)),duocarmycins, geldanamycins, methotrexates, thiotepa, vindesines,vincristines, hemiasterlins, nazumamides, microginins, radiosumins,topoisomerase I inhibitors, alterobactins, microsclerodermins,theonellamides, esperamicins, PNU-159682, and their analogues andderivatives above thereof.

7). Other immunotheraphy drugs: e.g. imiquimod, interferons (e.g. c,J3), granulocyte colony-stimulating factors, cytokines, Interleukins(IL-1˜IL-35), antibodies (e. g. trastuzumab, pertuzumab, bevacizumab,cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab,omalizumab, PD-1 or PD-L1), Protein-bound drugs (e.g., Abraxane), anantibody conjugated with drugs selected from calicheamicin derivative,of maytansine derivatives (DM1 and DM4), CC-1065, SN-38, exatecan,topotecan, topoisomerase I inhibitors, duocarmycin, PBD or IGN minorgroove binders, potent taxol derivatives, doxorubicin, auristatinantimitotic drugs (e. g. Trastuzumab-DM1, Trastuzumab deruxtecan(DS-8201a), Inotuzumab ozogamicin, Brentuximab vedotin, Sacituzumabgovitecan, Glembatumumab vedotin, lorvotuzumab mertansine, AN-152 LMB2,TP-38, VB4-845, Cantuzumab mertansine, AVE9633, SAR3419, CAT-8015(anti-CD22), IMGN388, Mirvetuximab soravtansine (IMGN853), Enfortumabvedotin, milatuzumab-doxorubicin, SGN-75 (anti-CD70),anti-Her3-exetecan, anti-Trop2-exetecan, nnti-CD79b-MMAE,anti-Her2-MMAE, anti-trop2-MMAE, anti-Her2-MMAF, anti-trop2-MMAF,anti-CD22-calicheamicin derivative, anti-CD22-MMAE, anti-Her2-auristatinderivatives, anti-Muc1-auristatin derivatives, anti-cMet-auristatinderivatives, or anti-Claudin 18.2-auristatin derivatives).

8). The pharmaceutically acceptable salts, acids or derivatives of anyof the above drugs.

In another synergistic immunotherapy, an antibody of a checkpointinhibitor, TCR (T cell receptors) T cells, or CARs (chimeric antigenreceptors) T cells, or of B cell receptor (BCR), Natural killer (NK)cells, or the cytotoxic cells, or an antibody of anti-CD3, CD4, CD8,CD16 (FcγRIII), CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38,CD40, CD40L, CD45RA, CD45RO, CD56, CD57, CD57^(bright), CD70, CD79,CD79b, CD123, CD125, CD138, TNFβ, Fas ligand, MHC class I molecules(HLA-A, B, C), VEGF, or NKR-Plantigen is preferred to use along with theconjugates of the present patent for synergistic therapy.

In yet another embodiment, a pharmaceutical composition comprising atherapeutically effective amount of the conjugate of Formula (I)˜(VII)or any conjugates described through the present patent can beadministered concurrently with the other therapeutic agents such as thechemotherapeutic agent, the radiation therapy, immunotherapy agents,autoimmune disorder agents, anti-infectious agents or the otherconjugates for synergistically effective treatment or prevention of acancer, or an autoimmune disease, or an infectious disease.

According to a still further object, the present invention is alsoconcerned with the process of preparation of the conjugate of theinvention. The conjugate and process of the present invention may beprepared in a number of ways well known to those skilled in the art. Theantimitotic agents used in the conjugate can be synthesized, forexample, by application or adaptation of the methods described below, orvariations thereon as appreciated by the skilled artisan. Theappropriate modifications and substitutions will be readily apparent andwell known or readily obtainable from the scientific literature to thoseskilled in the art. In particular, such methods can be found in R. C.Larock, Comprehensive Organic Transformations, 2^(nd) Edition, Wiley-VCHPublishers, 1999.

In the reactions described hereinafter, it may be necessary to protectreactive functional groups, for example hydroxy, amino, imino, thio orcarboxy groups, where these are desired in the final product, to avoidtheir unwanted participation in the reactions. Conventional protectinggroups may be used in accordance with standard practice, for examplessee P. G. Wuts and T. W. Greene, Greene's Protective Groups in OrganicSynthesis, Wiley-Interscience; 4th edition (2006). Some reactions may becarried out in the presence of a base, or an acid or in a suitablesolvent. There is no particular restriction on the nature of the base,acid and solvent to be used in this reaction, and any base, acid orsolvent conventionally used in reactions of this type may equally beused here, provided that it has no adverse effect on other parts of themolecule. The reactions can take place over a wide range oftemperatures. In general, we find it convenient to carry out thereaction at a temperature of from −80° C. to 150° C. (more preferablyfrom about room temperature to 100° C.). The time required for thereaction may also vary widely, depending on many factors, notably thereaction temperature and the nature of the reagents. However, providedthat the reaction is effected under the preferred conditions outlinedabove, a period of from 3 hours to 20 hours will usually suffice.

The work-up of the reaction can be carried out by conventional means.For example, the reaction products may be recovered by distilling offthe solvent from the reaction mixture or, if necessary after distillingoff the solvent from the reaction mixture, pouring the residue intowater followed by extraction with a water-immiscible organic solvent anddistilling off the solvent from the extract. Additionally, the productcan, if desired, be further purified by various well known techniques,such as recrystallization, reprecipitation or the various chromatographytechniques, notably column chromatography or preparative thin layerchromatography. The synthesis of the antimitotic agents and theirconjugates of this invention are illustrated in FIGS. 1-21 .

The conjugates of the cell-binding molecules with potent antimitoticagents are further illustrated but not restricted by the description inthe following examples.

EXPERIMENTAL MATERIALS

Mass spectra were obtained using a Bruker Esquire 3000 system. NMRspectra were recorded on a Bruker AVANCE300 spectrometer. Chemicalshifts are reported in ppm relative to TMS as an internal standard.Ultraviolet spectra were recorded on a Hitachi U1200 spectrophotometer.HPLC was performed using an Agilent 1100 HPLC system equipped with afraction collector and a variable wavelength detector. Thin layerchromatography was performed on Analtech GF silica gel TLC plates.Aminal acids and their derivatives as well as preloaded resins wereeither from Merck Chemicals International Co, or Synthetech Co., orPeptides International Inc or Chembridge International Co. orSigma-Aldrich Co. Some of the linkers, Linkers of NHS ester/Maleimide(AMAS, BMPS, GMBS, MBS, SMCC, EMCS or Sulfo-FMCS, SMPB, SMPH, LC-SMCC,Sulfo-KMUS, SM(PEG)4, SM(PEG)6, SM(PEG)8, SM(PEG)12, SM(PEG)24); NHSester/Pyridyldithiol (SPDP, LC-SPDP or Sulfo-LC-SPDP, SMPT,Sulfo-LC-SMPT); NHS esters/H-aloacetyl (SIA, SBAP, SIAB or Sulfo-SLAB);NHS ester/Diazirine (SDA or Sulfo-SDA, LC-SDA or Sulfo-LC-SDA, SDAD orSulfo-SDAD); Maleimide/Hydrazide (BMPH, EMCH, MPBH, KMUH);Pyridyldithiol/Hydrazide (PDPH); Isocyanate/Maleimide (PMPI) werepurchased from Thermo Fisher Scientific Co. SPDB, SPP linkers were madeaccording to the references (Cumber, A. et al, Bioconjugate Chem., 1992,3, 397-401). T-DM1 and Trastuzumab was from Genentech. All otherchemicals or anhydrous solvents were from Sigma-Aldrich International orAladdin Chemical (Shanghai) Ltd.

Example 1. Synthesis ofdi-tert-butyl-1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate

To di-tert-butyl hydrazine-1,2-dicarboxylate (8.01 g, 34.4 mmol) in DMF(150 ml) was added NaH (60% in oil, 2.76 g, 68.8 mmol). After stirred atRT for 30 min, tert-butyl 2-bromoacetate (14.01 g, 72.1 mmol) was added.The mixture was stirred overnight, quenched with addition of methanol (3ml), concentrated, diluted with EtOAc (100 ml) and water (100 ml),separated, and the aqueous layer was extracted with EtOAc (2×50 ml). Theorganic layers were combined, dried over MgSO₄, filtered, evaporated,and purified by SiO₂ column chromatography (EtOAc/Hexane 1:5 to 1:3) toafforded the title compound (12.98 g, 82% yield) as a colorless oil. MSESI m/z calcd for C₂₂H₄₁N₂O₈ [M+H]⁺ 461.28, found 461.40.

Example 2. Synthesis of 2,2′-(hydrazine-1,2-diyl)diacetic acid

Di-tert-butyl1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (6.51 g,14.14 mmol) in 1,4-dioxane (40 ml) was added HCl (12 M, 10 ml). Themixture was stirred for 30 min, diluted with dioxane (20 ml) and toluene(40 ml), evaporated and co-evaporated with dioxane (20 ml) and toluene(40 ml) to dryness to afford the crude title product for the next stepwithout further production (2.15 g, 103% yield, ˜93% pure). MS ESI m/zcalcd for C₄H₉N₂O₄ [M+H]⁺ 149.05, found 149.40.

Example 3. Synthesis of2,2′-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetic acid

To a solution of 2,2′-(hydrazine-1,2-diyl)diacetic acid (1.10 g, 7.43mmol) in the mixture of THF (50 ml) and NaH₂PO₄ (0.1 M, 80 ml, pH 6.0)was added (E)-3-bromoacryloyl bromide (5.01 g, 23.60 mmol). The mixturewas stirred for 6 h, concentrated and purified on SiO₂ column elutedwith H₂O/CH₃CN (1:9) containing 3% formic acid to afford the titlecompound (2.35 g, 77% yield, ˜93% pure). MS ESI m/z calcd forC₁₀H₁₁Br₂N₂O₆ [M+H]⁺ 412.89, found 413.50.

Example 4. Synthesis of2,2′-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetyl chloride

2,2′-(1,2-Bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetic acid (210mg, 0.509 mmol) in dichloroethane (15 ml) was added (COCl)₂ (505 mg,4.01 mmol), followed by addition of 0.040 ml of DMF. After stirred at RTfor 2 h, the mixture was concentrated and co-evaporated withdichloroethane (2×20 ml) and toluene (2×15 ml) to dryness to afford thetitle crude product (which is not stable) for the next step withoutfurther purification (245 mg, 107% yield). MS ESI m/z calcd forC10H₉Br₂Cl₂N₂O₄ [M+H]⁺ 448.82, 450.82, 452.82, 454.82, found 448.60,450.60, 452.60, 454.60.

Example 5. Synthesis of tert-butyl 2,8-dioxo-1,5-oxazocane-5-carboxylate

To a solution of 3,3′-azanediyldipropanoic acid (10.00 g, 62.08 mmol) in1.0 M NaOH (300 ml) at 4° C. was added di-tert-butyl dicarbonate (22.10g, 101.3 mmol) in 200 ml THF in 1 h. After addition, the mixture waskept to stirring for 2 h at 4° C. The mixture was carefully acidified topH˜4 with 0.2 M H₃PO₄, concentrated in vacuo, extracted with CH₂Cl₂,dried over Na₂SO₄, evaporated and purified with flash SiO₂chromatography eluted with AcOH/MeOH/CH₂Cl₂ (0.01:1:5) to afford3,3′-((tert-butoxycarbonyl)azanediyl)dipropanoic acid (13.62 g, 84%yield). ESI MS m/z C₁₁H₁₉NO₆ [M+H]⁺, calcd. 262.27, found 262.40.

To a solution of 3,3′-((tert-butoxycarbonyl)azanediyl)dipropanoic acid(8.0 g, 30.6 mmol) in CH₂Cl₂ (500 ml) at 0° C. was added phosphoruspentoxide (8.70 g, 61.30 mmol). The mixture was stirred at 0° C. for 2 hand then r.t. for 1 h, filtered through short SiO₂ column, and rinsedthe column with EtOAc/CH₂Cl₂ (1:6). The filtrate was concentrated andtriturated with EtOAc/hexane to afford the title compound (5.64 g, 74%yield). ESI MS m/z C₁₁H₁₇NO₅ [M+H]⁺, calcd. 244.11, found 244.30.

Example 6. Synthesis of 2,5-dioxopyrrolidin-1-yl propiolate

Propiolic acid (5.00 g, 71.4 mmol), NHS (9.01 g, 78.3 mmol) and EDC(20.0 g, 104.1 mmol) in CH₂Cl₂ (150 ml) and DIPEA (5 ml, 28.7 mmol) wasstirred for overnight, evaporated and purified by SiO₂ columnchromatography (EtOAc/Hexane 1:4) to afforded the title compound (9.30g, 79% yield) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 2.68 (s,1H), 2.61 (s, 4H). MS ESI m/z calcd for C₇H₅NaNO₄ [M+Na]⁺ 190.02, found190.20.

Example 7. Synthesis of tert-butyl 2-propioloylhydrazinecarboxylate

Propiolic acid (5.00 g, 71.4 mmol), tert-butyl hydrazinecarboxylate(9.45 g, 71.5 mmol) and EDC (20.0 g, 104.1 mmol) in CH₂Cl₂ (150 ml) andDIPEA (5 ml, 28.7 mmol) was stirred for overnight, evaporated andpurified by SiO₂ column chromatography (EtOAc/Hexane 1:5) to affordedthe title compound (7.92 g, 84% yield) as a colorless oil. ¹H NMR (500MHz, CDCl₃) δ 8.76 (m, 2H), 2.68 (s, 1H), 1.39 (s, 9H). MS ESI m/z calcdfor C₅H₁₂NaN₂O₂ [M+Na]⁺ 155.09, found 155.26.

Example 8. Synthesis of propiolohydrazide, HCl salt

tert-butyl 2-propioloylhydrazinecarboxylate (4.01 g, 30.35 mmol)dissolved in 1,4-dioxane (12 mL) was treated with 4 ml of HCl (conc.) at4° C. The mixture was stirred for 30 min, diluted with Dioxane (30 ml)and toluene (30 ml) and concentrated under vacuum. The crude mixture waspurified on silica gel using a mixture of methanol (from 5% to 10%) and1% formic acid in methylene chloride as the eluant to give titlecompound (2.11 g, 83% yield), ESI MS m/z C₃H₅N₂O [M+H]⁺, calcd. 85.03,found 85.30.

Example 9. Synthesis of Compound 2

In a 10-L reactor 2,2-diethoxyacetonitrile (1.00 kg, 7.74 mol, 1.0 eq.)was mixed with (NH₄)₂S (48% aqueous solution, 1.41 kg, 9.29 mol, 1.2eq.) in methanol (6.0 L) at room temperature. The internal temperatureincreased to 33° C. and then dropped back to r.t. After stirringovernight, the reaction mixture was concentrated under vacuum and theresidue was taken up in ethyl acetate (5 L) and washed with saturatedNaHCO₃ solution (4×1.0 L). The aqueous layer was back-extracted withethyl acetate (5×1.0 L). The organic phases were combined and washedwith brine (3 L), dried over anhydrous Na₂SO₄ and concentrated. Theresulting solid was collected by vacuum filtration and washed withpetroleum ether. The filtrate was concentrated and triturated withpetroleum ether to yield a few crops of white or light yellow solid. Allcrops were combined to give 1.1 kg of desired product (87% yields). ¹HNMR (500 MHz, CDCl₃) δ 7.81 (d, J=71.1 Hz, 2H), 5.03 (s, 1H), 3.73 (dq,J=9.4, 7.1 Hz, 2H), 3.64 (dq, J=9.4, 7.0 Hz, 2H), 1.25 (t, J=7.1 Hz,6H).

Example 10. Synthesis of Compound 3

In a 5-L 3-neck round bottle flask, equipped with a reflux condenser andan additional funnel, ethyl bromopyruvate (80% purity, 404 mL, 2.57 mol,1.2 eq.) was added over 30 min. to a mixture of molecular sieves (3 Å,500 g) and thioamide (350 g, 2.14 mol, 1.0 eq.) in 3 L EtOH. Duringaddition, the internal temperature increased slightly. The reactionmixture was then heated to reflux and stirred for 30 min. After coolingto r.t. the reaction mixture was filter over celite and the filter cakewashed with ethyl acetate. The filtrate was concentrated under vacuum.Two batches of the crude product were combined and mixed with silica gel(1.5 kg) and loaded on a silica gel (10 kg packed) column and elutedwith ethyl acetate/petroleum ether (10-20%) to give thiazole carboxylateas a brown oil (509 g, 92% yield).

Example 11. Synthesis of Compound 4

A solution of acetal (300 g, 1.16 mol) in acetone (3.0 L) was heated toreflux and 4N HCl (250 mL) was added over 1.0 h to the refluxingsolution. TLC analysis indicated complete consumption of the startingmaterial. The reaction mixture was concentrated under reduced pressureand phases were separated. The organic phase was diluted with ethylacetate (1.5 L) and washed with saturated NaHCO₃ solution (1.0 L), water(1.0 L) and brine (1.0 L), and then dried over anhydrous Na₂SO₄. All ofthe aqueous phases were combined and extracted with ethyl acetate. Theextracts were combined and dried over anhydrous Na₂SO₄. The organicsolutions were filtered and concentrated under reduced pressure. Thecrude product was triturated with petroleum ether and diethyl ether(5:1) and the resulting solid was collected by vacuum filtration andwashed with petroleum ether and ethyl acetate (10:1). The filtrate wasconcentrated and chromatographed using 0-15% ethyl acetate/petroleumether to give another crop of desired product. All white to light yellowsolids were combined and weighed 40 g (43% yield). ¹H NMR (500 MHz,CDCl₃) δ 10.08-10.06 (m, 1H), 8.53-8.50 (m, 1H), 4.49 (q, J=7.1 Hz, 2H),1.44 (t, J=7.1 Hz, 3H). MS ESI m/z calcd for C₇H₈NO₃S [M+H]⁺ 186.01;found 186.01.

Example 12. Synthesis of Compound 6

NaN₃ (740 g, 11.4 mol) was dissolved in water (2.0 L) anddichloromethane (2.0 L) was added and cooled at 0° C., to which Tf₂O(700 mL, 4.10 mol, 1.8 eq.) was added over 1.5 h. After addition wascompleted, the reaction was stirred at 0° C. for 3 h. The organic phasewas separated and the aqueous phase was extracted with dichloromethane(2×500 mL). The combined organic phases were washed with saturatedNaHCO₃ solution (3×1.0 L). This dichloromethane solution of triflylazide was added to a mixture of (L)-isoleucine (300 g, 2.28 mol, 1.0eq.), K₂CO₃ (472 g, 3.42 mol, 1.5 eq.), CuSO₄·5H₂O (5.7 g, 22.8 mmol,0.01 eq.) in water (3.0 L) and methanol (3.0 L) at r.t. During addition,the internal temperature increased slightly. And the mixture was thenstirred at r.t. for 16 h. The organic solvents were removed underreduced pressure and the aqueous phase was acidified to pH 6-6.5 withconcentrated HCl (about 280 mL added) and then diluted with phosphatebuffer (0.25 M, pH 6.2, 6.0 L), washed with EtOAc (6×2.0 L) to removethe sulfonamide by-product. The solution was acidified to pH 3 withconcentrated HCl (about 400 mL added), extracted with EtOAc (4×2.0 L).The combined organic layers were washed with brine (2.0 L) and driedover anhydrous Na₂SO₄, filtered and concentrated to give product 6 (320g, 89% yield) as a light yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 12.01 (s,1H), 3.82 (d, J=5.9 Hz, 1H), 2.00 (ddd, J=10.6, 8.6, 5.5 Hz, 1H), 1.54(dqd, J=14.8, 7.5, 4.4 Hz, 1H), 1.36-1.24 (m, 1H), 1.08-0.99 (m, 3H),0.97-0.87 (m, 3H).

Example 13. Synthesis of Compound 10

To a solution of (S)-2-methylpropane-2-sulfinamide (100 g, 0.825 mol,1.0 eq.) in 1 L THF was added Ti(OEt)₄ (345 mL, 1.82 mol, 2.2 eq.) and3-methyl-2-butanone (81 mL, 0.825 mol, 1.0 eq.) under N₂ at r.t. Thereaction mixture was refluxed for 16 h, then cooled to r.t. and pouredonto iced water (1 L). The mixture was filtered and the filter cake waswashed with EtOAc. The organic layer was separated, dried over anhydrousNa₂SO₄ and concentrated to give a residue which was purified by vacuumdistillation (15-20 torr, 95° C.) to afforded product 10 (141 g, 90%yield) as a yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 2.54-2.44 (m, 1H),2.25 (s, 3H), 1.17 (s, 9H), 1.06 (dd, J=6.9, 5.1 Hz, 6H). MS ESI m/zcalcd for C₉H₁₉NaNOS [M+Na]⁺ 212.12; found 212.11.

Example 14. Synthesis of Compound 11

To a solution of diisopropylamine (264 mL, 1.87 mol, 1.65 eq.) in dryTHF (1 L) was added n-butyllithium (2.5 M, 681 mL, 1.70 mol, 1.5 eq.) at−78° C. under N₂. The reaction mixture was warmed to 0° C. over 30 minand then cooled back to −78°. Compound 10 (258 g, 1.36 mol, 1.2 eq.) wasadded, and rinsed with THF (50 mL). The reaction mixture was stirred for1 h before ClTi(O^(i)Pr)₃ (834 g, 3.17 mol, 2.8 eq.) in THF (1.05 L) wasadded dropwise. After stirring for 1 h, compound 4 (210 g, 1.13 mol, 1.0eq.) dissolved in THF (500 mL) was added dropwise in about 1 hours andthe resulting reaction mixture was stirred for 3 h. The completion ofthe reaction was indicated by TLC analysis. The reaction was quenched bya mixture of acetic acid and THF (v/v 1:1, 300 mL), then poured ontobrine (2 L), extracted with EtOAc (8×1 L). The organic phase was washedwith water and brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The residue was purified by column chromatography(DCM/EtOAc/PE 2:1:2) to afforded the compound 11 (298 g, 74% yield) as acolorless oil. ¹H NMR (500 MHz, CDCl₃) δ 8.13 (s, 1H), 6.63 (d, J=8.2Hz, 1H), 5.20-5.11 (m, 1H), 4.43 (q, J=7.0 Hz, 2H), 3.42-3.28 (m, 2H),2.89 (dt, J=13.1, 6.5 Hz, 1H), 1.42 (t, J=7.1 Hz, 3H), 1.33 (s, 9H),1.25-1.22 (m, 6H). MS ESI m/z calcd for C₁₆H₂₆NaN₂O₄S₂ [M+Na]⁺ 397.13,found 397.11.

Example 15. Synthesis of Compound 12

A solution of compound 11 (509 g, 1.35 mol, 1.0 eq.) dissolved in THF(200 mL) was cooled to −78° C. Ti(OEt)₄ (570 mL, 2.72 mol, 2.0 eq.) wasadded slowly. After completion of the addition, the mixture was stirredfor 1 h, before NaBH₄ (51.3 g, 1.36 mol, 1.0 eq.) was added in portionsover 90 min. The reaction mixture was stirred at −78° C. for 3 h. TLCanalysis showed starting material still remained. EtOH (50 mL) was addedslowly, and the reaction was stirred for 1.5 h and then poured ontobrine (2 L, with 250 mL HOAc) and warmed to r.t. After filtration overCelite, the organic phase was separated and washed with water and brine,dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue waspurified by column chromatography (EtOAc/PE 1:1) to deliver product 12(364 g, 71% yield) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.10 (s,1H), 5.51 (d, J=5.8 Hz, 1H), 5.23-5.15 (m, 1H), 4.41 (q, J=7.0 Hz, 2H),3.48-3.40 (m, 1H), 3.37 (d, J=8.3 Hz, 1H), 2.29 (t, J=13.0 Hz, 1H),1.95-1.87 (m, 1H), 1.73-1.67 (m, 1H), 1.40 (t, J=7.1 Hz, 3H), 1.29 (s,9H), 0.93 (d, J=7.3 Hz, 3H), 0.90 (d, J=7.2 Hz, 3H). MS ESI m/z calcdfor C₁₆H₂₈NaN₂O₄S₂ [M+Na]⁺ 399.15, found 399.14.

Example 16. Synthesis of Compound 13

To a solution of compound 12 (600 g, 1.60 mol, 1.0 eq.) in ethanol (590mL) was added 4 N HCl in dioxane (590 mL) slowly at 0° C. The reactionwas allowed to warm to r.t. and stirred for 2.5 h. A white precipitatecrushed out and was collected by filtration and washed with EtOAc. Thefiltrate was concentrated and triturated with EtOAc. Two crops of whitesolid were combined and weighed 446 g (90% yield).

Example 17. Synthesis of Compound 14

Compound 10: Azido-Ile-OH (6, 153 g, 0.97 mol, 2.0 eq.) was dissolved inTHF (1.5 L) and cooled to 0° C., to which NMM (214 mL, 1.94 mol, 4.0eq.) and isobutylchloroformate (95 mL, 0.73 mol, 2.0 eq.) were added insequence. The reaction was stirred at 0° C. for 1.0 h. Compound 13 (150g, 0.49 mmol, 1.0 eq.) was added in portions. After stirring at 0° C.for 30 min, the reaction was warmed to r.t. and stirred for 2 h. Waterwas added at 0° C. to quench the reaction and the resulting mixture wasextracted with EtOAc for three times. The combined organic layers werewashed with 1N HCl, saturated NaHCO₃ and brine, dried over anhydrousNa₂SO₄, filtered and concentrated. The residue was purified by columnchromatography (0-30% EtOAc/PE) to give a white solid (140 g, 70%yield). ¹H NMR (500 MHz, CDCl₃) δ 8.14 (s, 1H), 6.57 (d, J=8.9 Hz, 1H),4.91 (d, J=11.1 Hz, 1H), 4.44 (dd, J=13.2, 6.3 Hz, 2H), 4.08-3.95 (m,2H), 2.21 (dd, J=24.4, 11.5 Hz, 2H), 1.90-1.79 (m, 3H), 1.42 (t, J=6.6Hz, 3H), 1.37-1.27 (m, 2H), 1.11 (d, J=6.4 Hz, 3H), 1.01-0.94 (m, 9H).MS ESI m/z calcd for C18H₃₀N₅O₄S [M+H]⁺ 412.19, found 412.19.

Example 18. Synthesis of Compound 15

Compound 11: To a solution of compound 14 (436 g, 1.05 mol, 1.0 eq.) inCH₂Cl₂ (50 mL) was added imidazole (94 g, 1.37 mmol, 1.3 eq.), followedby chlorotriethylsilane (222 mL, 1.32 mol, 1.25 eq.) at 0° C. Thereaction mixture was allowed to warm to r.t. over 1 hour and stirred foran additional hour. Brine was added to the reaction mixture, the organiclayer was separated and the aqueous layer was extracted with EtOAc. Thecombined organic phases were dried, filtered, concentrated under reducedpressure, and purified by column chromatography with a gradient of15-35% EtOAc in petroleum ether to afford product 15 (557.4 g, 95%yield) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 8.12 (s, 1H), 6.75(d, J=8.0 Hz, 1H), 5.20-5.12 (m, 1H), 4.44 (q, J=7.0 Hz, 2H), 4.06-3.97(m, 1H), 3.87 (d, J=3.8 Hz, 1H), 2.14 (d, J=3.8 Hz, 1H), 2.01-1.91 (m,3H), 1.42 (t, J=7.1 Hz, 3H), 1.34-1.25 (m, 2H), 1.06 (d, J=6.8 Hz, 3H),1.00-0.93 (m, 18H), 0.88 (dd, J=19.1, 6.8 Hz, 6H). MS ESI m/z calcd forC₂₄H₄₄N₅O₄SSi [M+H]+ 526.28, found 526.28.

Example 19. Synthesis of Compound 16

To a solution of 15 (408 g, 0.77 mol, 1.0 eq.) and methyl iodide (145mL, 2.32 mol, 3.0 eq.) in THF (4 L) was added sodium hydride (60%dispersion in mineral oil, 62.2 g, 1.55 mol, 2.0 eq.) at 0° C. Theresulting mixture was stirred at 0° C. overnight and then poured ontoice-water cooled saturated ammonium chloride (5 L) with vigorousstirring. The mixture was then extracted with EtOAc (3×500 mL) and theorganic layers were dried, filtered, concentrated and purified by columnchromatography with a gradient of 15-35% EtOAc in petroleum ether toafford product 16 (388 g, 93% yield) as a light yellow oil. ¹H NMR (500MHz, CDCl₃) δ 8.09 (s, 1H), 4.95 (d, J=6.6 Hz, 1H), 4.41 (q, J=7.1 Hz,2H), 3.56 (d, J=9.5 Hz, 1H), 2.98 (s, 3H), 2.27-2.06 (m, 4H), 1.83-1.70(m, 2H), 1.41 (t, J=7.2 Hz, 3H), 1.29 (ddd, J=8.9, 6.8, 1.6 Hz, 3H),1.01 (d, J=6.6 Hz, 3H), 0.96 (dt, J=8.0, 2.9 Hz, 15H), 0.92 (d, J=6.6Hz, 3H), 0.90 (d, J=6.7 Hz, 3H). MS ESI m/z calcd for C₂₅H₄₆N₅O₄SSi[M+H]⁺ 540.30, found 540.30.

Example 20. Synthesis of Compound 17

To a solution of compound 16 (1.01 g, 1.87 mmol) in methanol (15 mL) wasadded 0.1N HCl dropwise until a neutral pH was reached. After additionof Pd/C (10 wt %, 583 mg), the mixture was stirred under H₂ (1 atm) atroom temperature for 16 h. The Pd/C was then removed by filtration, withwashing of the filter pad with methanol. The filtrate was concentratedunder reduced pressure and the residue was re-dissolved in EtOAc (50mL), dried over anhydrous Na₂SO₄, filtered and concentrated to affordcompound 17 (900 mg, 94% yield) as a pale yellow oil.

Example 21. Synthesis of Compound 22

To a solution of D-pipecolinic acid (10.0 g, 77.4 mmol, 1.0 eq.) inmethanol (100 mL) was added formaldehyde (37% aqueous solution, 30.8 mL,154.8 mmol, 2.0 eq.), followed by Pd/C (10 wt %, 1.0 g). The reactionmixture was stirred under H₂ (1 atm) overnight, and then filteredthrough Celite, with washing of the filter pad with methanol. Thefiltrate was concentrated under reduced pressure to afford compound 22(10.0 g, 90% yield) as a white solid.

Example 22. Synthesis of Compound 23

To a solution of D-N-methyl pipecolinic acid (2.65 g, 18.5 mmol) inEtOAc (50 mL) were added pentafluorophenol (3.75 g, 20.4 mmol) and DCC(4.21 g, 20.4 mmol). The reaction mixture was stirred at r.t. for 16 h,and then filtered over Celite. The filter pad was washed with 10 mL ofEtOAc. The filtrate was used immediately without further purification orconcentration.

Example 23. Synthesis of Compound 28

A mixture of 2-amino-2-methylpropanoic acid (500 g, 4.85 mol, 1.0 eq.),aqueous formaldehyde (37%, 1.0 L, 12.1 mol, 2.5 eq.) and formic acid(1.0 L) was heated to reflux (80° C.) for 3.0 h. 6 N HCl (850 mL) wasthen added at r.t. and the reaction mixture was concentrated. Theresulting solid was collected by filtration with washing of ethylacetate for three times (1.0 L). The solid was dissolved in water (1.5L) and neutralized to pH 7.0 with 4N NaOH (about 1.0 L solution). Thesolution was concentrated and co-evaporated with ethanol (2.0 L) toremove residual water. MeOH (2.0 L) was added to the residue and thesolid (NaCl) was filtered off with washing of ethyl acetate. Thefiltrate was concentrated under reduced pressure to give a white solid639.2 g, which contains some NaCl and was used without furthertreatment.

Example 24. Synthesis of Compound 29

To a solution of 2-(dimethylamino)-2-methylpropanoic acid (97 g, 0.74mol) in EtOAc (1 L) were added pentafluorophenol (163 g, 0.88 mol) andDIC (126 mL, 0.81 mol). The reaction mixture was stirred at r.t. for 24h, and then filtered over Celite. The filter pad was washed with 10 mLof EtOAc. The filtrate was used immediately without further purificationor concentration.

Example 25. Synthesis of Compound 30

Dry Pd/C (10 wt %, 300 mg) and azide compound 16 (3.33 g, 6.61 mmol)were added to pentafluorophenyl ester 23 in EtOAc. The reaction mixturewas stirred under hydrogen atmosphere for 27 h, and then filteredthrough a plug of Celite, with washing of the filter pad with EtOAc. Thecombined organic portions were concentrated and purified by columnchromatography with a gradient of 0-5% methanol in EtOAc to delivercompound 30 (3.90 g, 86% yield). MS ESI m/z calcd for C₃₂H₅₉N₄O₅SSi[M+H]⁺ 639.39, found 639.39.

Example 26. Synthesis of Compound 31

The coupled product compound 30 (3.90 g, 6.1 mmol) was dissolved inAcOH/water/THF (v/v/v 3:1:1, 100 mL), and stirred at r.t. for 48 h. Thereaction was then concentrated and purified by column chromatography(2:98 to 15:85 MeOH/EtOAc) to afford compound 31 (2.50 g, 72% yield over2 steps). MS ESI m/z calcd for C₂₆H₄₅N₄O₅S [M+H]⁺ 525.30, found 525.33.

Example 27. Synthesis of Compound 32

An aqueous solution of LiOH (0.4 N, 47.7 mL, 19.1 mmol, 4.0 eq.) wasadded to a solution of compound 31 (2.50 g, 4.76 mmol, 1.0 eq.) indioxane (47.7 mL) at 0° C. The reaction mixture was stirred at r.t. for2 h and then concentrated. Column chromatography (100% CH₂Cl₂ toCH₂Cl₂/MeOH/NH₄OH 80:20:1) afforded compound 32 (2.36 g, 99% yield) asan amorphous solid. MS ESI m/z calcd for C₂₄H₄₁N₄O₅S [M+H]⁺ 497.27,found 497.28.

Example 28. Synthesis of Compound 33

To a solution of compound 32 (2.36 g, 4.75 mmol) in pyridine (50 mL) at0° C., acetic anhydride (2.25 mL, 24 mmol) was added slowly. Thereaction mixture was allowed to warm to r.t. over 2 h and stirred atr.t. for 24 h. The reaction was concentrated and then treated withdioxane/water (v/v 1:1, 10 mL) for 1 h to destroy possible anhydride.After concentration the residue was purified by column chromatography(100% CH₂Cl₂ to CH₂Cl₂/MeOH/NH₄OH 50:50:1) to afford compound 33 (2.25g, 88% yield) as an amorphous white solid. MS ESI m/z calcd forC₂₆H₄₃N₄O₆S [M+H]⁺ 539.28, found 539.28.

Example 29. Synthesis of Compound 38

To the EtOAc solution of pentafluorophenyl ester 29, compound 16 (200 g,0.37 mol) and dry Pd/C (10 wt %, 10 g) were added. The reaction mixturewas stirred under hydrogen atmosphere (1 atm) for 27 h, and thenfiltered through a plug of Celite, with washing of the filter pad withEtOAc. The combined organic portions were concentrated and purified bycolumn chromatography with a gradient of 0-5% methanol in EtOAc todeliver compound 38 (184 g, 79% yield). MS ESI m/z calcd forC₃₁H₅₈N₄O₅SSi [M+H]⁺ 627.39, found 627.39.

Example 30. Synthesis of Compound 39

Compound 38 (200 g, 0.32 mmol) was dissolved in AcOH/water/THF (v/v/v3:1:1, 638 mL), and stirred at r.t. for 4 days. After the reaction wasconcentrated, toluene was added and concentrated again; this step wasrepeated two times to afford compound 39, which was used directly in thenext step. MS ESI m/z calcd for C₂₅H₄₅N₄O₅S [M+H]⁺ 513.30, found 513.30.

Example 31. Synthesis of Compound 40

An aqueous solution of LiOH (0.4 N, 600 mL, 2.55 mol, 8.0 eq.) was addedto a solution of compound 39 (160 g, 0.319 mol, 1.0 eq.) in MeOH (1.2 L)at 0° C. The reaction mixture was stirred at r.t. for 2 h and thenconcentrated. Column chromatography (pure CH₂Cl₂ to 80:20:1CH₂Cl₂/MeOH/NH₄OH) afforded compound 40 (140 g, 91% yield for two steps)as an amorphous solid. MS ESI m/z calcd for C₂₃H₄₀N₄O₅S [M+H]⁺ 485.27,found 485.27.

Example 32. Synthesis of Compound 41

A solution of compound 27 (143 g, 0.30 mol, 1.0 eq.) and DMAP (0.36 g,2.95 mmol, 0.01 eq.) in anhydrous THF (1.4 L) and anhydrous DMF (75 mL)was cooled to 0° C., to which TEA (82.2 mL, 0.59 mmol, 2.0 eq.) andacetic anhydride (56 mL, 0.59 mmol, 2.0 eq.) were added. The reactionmixture was allowed to warm to r.t. and stirred for 24 h, and thenconcentrated. Column chromatography (5-50% MeOH/DCM) delivered compound41 (147 g, 95% yield) as an amorphous solid. MS ESI m/z calcd forC₂₅H₄₄N₄O₆S [M+H]⁺ 527.28, found 527.28.

Example 33. Synthesis of Compound 41a

To a solution of compound 41 (5.0 g, 9.5 mmol, 1.0 eq) in anhydrous DCM(100 mL) was added EDC (4.6 g, 23.8 mmol, 2.5 eq) and pentafluorophenol(4.4 g, 23.8 mmol, 2.5 eq) at room temperature under N₂. The mixture wasstirred at room temperature for 2 h, and then diluted in DCM (100 mL),washed with water (2×200 mL) and brine (200 mL), dried over anhydroussodium sulfate, filtered, concentrated and purified by SiO₂ columnchromatography (50% EtOAc/PE) to give compound 41a as a white solid (5.2g, 79% yield) MS ESI m/z calcd for C₃₁H₄₂F₅N₄O₆S [M+H]⁺: 693.27, found:693.27.

Example 34. Synthesis of Compound 95

In a 500 mL round-bottomed flask equipped with a magnetic stir bar wasadded triphenylphosphine (100 g, 381 mmol, 1.0 eq.) and ethyl2-bromopropionate (100 mL, 762 mmol, 2.0 eq.). The mixture was thenheated to 50° C. under N₂ atmosphere overnight. After the white solid(PPh₃) was dissolved, a large amount of white solid was generated.Trituration with petroleum ether/EtOAc and filtration gave compound 95as a white solid (135 g, 80% yield). MS ESI m/z calcd for C₂₃H₂₄O₂P[M-Br]⁺ 363.15, found 363.13.

Example 35. Synthesis of Compound 96

A solution of compound 95 (135.42 g, 305.7 mmol) in dichloromethane (500mL) was added slowly into 10% NaOH solution (450 mL) with vigorousstirring. The organic solution rapidly turned bright yellow. After 30minutes, TLC analysis showed that the reaction was completed. Layerswere separated and the aqueous layer was further extracted with CH₂Cl₂(2×200 mL). Combined organic layers were washed with brine, dried overanhydrous Na₂SO₄ and concentrated to give a yellow solid 96 (104 g, 94%yield). MS ESI m/z calcd for C₂₃H₂₄O₂P [M+H]⁺ 362.14, found 363.13. Thecrude product was used directly in the next step.

Example 36. Synthesis of Compound 98

To a mixture of Boc-L-Tyr-OMe (670 g, 2.27 mol, 1.0 eq.), K₂CO₃ (358 g,2.5 mol, 1.1 eq.) and KI (38 g, 0.227 mol, 0.1 eq.) in acetone (3 L) wasadded benzyl bromide (283 mL, 2.38 mol, 1.05 eq.) slowly. The mixturewas then refluxed overnight. Water (6 L) was added and the reactionmixture was extracted with EtOAc (5×100 L). The combined organic layerswere washed with brine (2 L), dried over anhydrous Na₂SO₄, filtered,concentrated and purified by SiO₂ column chromatography (4:1hexanes/EtOAc) to give a white solid 98 (795 g, 91% yield). ¹H NMR (500MHz, CDCl₃) δ 7.43 (d, J=7.0 Hz, 2H), 7.38 (t, J=7.4 Hz, 2H), 7.32 (t,J=7.2 Hz, 1H), 7.04 (d, J=8.5 Hz, 2H), 6.91 (d, J=8.6 Hz, 2H), 5.04 (s,2H), 4.55 (d, J=6.9 Hz, 1H), 3.71 (s, 3H), 3.03 (qd, J=14.0, 5.8 Hz,2H), 1.43 (s, 9H). ESI: m/z: calcd for C₂₂H₂₈NO₅ [M+H]⁺: 386.19, found386.19.

Example 37. Synthesis of Compound 99

To a solution of ester 98 (380 g, 987 mmol, 1.0 eq.) in anhydrousdichloromethane (1 L) at −78° C. was added DIBAL (1.0 M in hexanes, 2.9L, 2.9 eq.) over 3 h. After the addition was completed, the mixture wasquenched with 3 L of ethanol. 1N HCl was added dropwise until pH 4 wasreached. The resulting mixture was allowed to warm to 0° C. Layers wereseparated and the aqueous layer was further extracted with EtOAc (3×3L). The combined organic solution was washed with brine, dried overanhydrous Na₂SO₄, and concentrated. Trituration with PE/EtOAc andfiltration gave a white solid 99 (263 g, 75% yield). ¹H NMR (500 MHz,CDCl₃) δ 9.65 (s, 1H), 7.45 (d, J=7.1 Hz, 2H), 7.41 (t, J=7.4 Hz, 2H),7.35 (t, J=7.1 Hz, 1H), 7.11 (d, J=8.6 Hz, 2H), 6.95 (d, J=8.6 Hz, 2H),5.07 (s, 2H), 4.42 (dd, J=12.4, 6.1 Hz, 1H), 3.09 (d, J=6.2 Hz, 2H),1.46 (s, 9H). ESI: m/z: calcd for C₂₁H₂₆NO₄ [M+H]⁺: 356.18, found356.19.

Example 38. Synthesis of Compound 100

To a solution of aldehyde 99 (81.4 g, 229 mmol, 1.0 eq.) in anhydrousdichloromethane (800 mL) at room temperature was added ylide 96 (2.0eq.) in anhydrous dichloromethane (800 mL) over 30 min. The mixture wasstirred at room temperature overnight then concentrated and purified bySiO₂ column chromatography (6:1 petroleum ether/EtOAc) to give a whitesolid 100 (63.4 g, 63% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.45-7.41 (m,2H), 7.40-7.35 (m, 2H), 7.33 (d, J=7.2 Hz, 1H), 7.10-7.06 (m, 2H),6.92-6.88 (m, 2H), 6.50 (dd, J=8.8, 1.3 Hz, 1H), 5.04 (s, 2H), 4.57 (s,2H), 4.18 (q, J=7.1 Hz, 2H), 2.86 (d, J=8.5 Hz, 1H), 2.72 (dd, J=13.6,6.8 Hz, 1H), 1.71 (d, J=1.4 Hz, 3H), 1.41 (d, J=2.2 Hz, 9H), 1.28 (td,J=7.5, 5.1 Hz, 4H). MS ESI m/z calcd for C₂₆H₃₃NaNO₅ [M+Na]⁺ 462.24,found 462.22.

Example 39. Synthesis of Compound 101

In a hydrogenation bottle, Pd/C (1.83 g, 10 wt %, 50% water) was addedto a solution of compound 100 (30.2 g, 68.9 mmol) in THF (100 mL) andmethanol (300 mL). The mixture was shaken under 1 atm H₂ overnight,filtered through Celite (filter aid), and the filtrate was concentratedto afford compound 101 (25.0 g, theoretical yield) as a colorless oil.¹H NMR (500 MHz, CDCl₃) δ 6.99 (d, J=7.0 Hz, 2H), 6.72 (d, J=7.6 Hz,2H), 4.39 (s, 1H), 4.18-4.04 (m, 2H), 3.82 (s, 1H), 2.60 (dd, J=37.2,20.9 Hz, 4H), 1.95-1.81 (m, 1H), 1.39 (s, 11H), 1.24 (dd, J=9.5, 4.3 Hz,3H), 1.13 (t, J=8.9 Hz, 3H). MS ESI m/z calcd for C₁₉H₃₁NO₅ [M+H]⁺352.20, found 352.19.

Example 40. Synthesis of Compound 102

To a solution of compound 101 (5.96 g, 35.9 mmol, 1.0 eq.) in anhydrousdichloromethane (200 mL) was added Ac₂O (3.2 mL, 33.9 mmol, 2.0 eq.) andHNO₃ (65%-68%, 3.5 mL, 50.79 mmol, 3.0 eq.) at room temperature. Themixture was stirred at room temperature for 30 min, and TLC analysisshowed that the reaction was completed. The reaction solution was washedwith water (3×200 mL), and the aqueous layer was back-extracted withdichloromethane (3×100 mL). The combined dichloromethane solution waswashed with brine, dried over anhydrous Na₂SO₄, filtered, concentratedand purified by SiO₂ column chromatography (5:1 hexanes/EtOAc) to givecompound 102 as a yellow solid (4.18 g, 72% yield). ¹H NMR (500 MHz,CDCl₃) δ 10.49 (s, 1H), 7.89 (s, 1H), 7.44 (d, J=8.4 Hz, 1H), 7.09 (d,J=8.6 Hz, 1H), 4.32 (d, J=8.3 Hz, 1H), 4.12 (dd, J=14.0, 7.0 Hz, 2H),3.80 (s, 1H), 2.76 (dd, J=13.0, 6.8 Hz, 2H), 2.59 (s, 1H), 1.88 (s, 1H),1.37 (t, J=8.7 Hz, 9H), 1.25 (dd, J=13.5, 6.9 Hz, 4H), 1.16 (t, J=8.0Hz, 3H). MS ESI m/z calcd for C₁₉H₂₈NaN₂O₇ [M+Na]⁺ 419.19, found 419.17.

Example 41. Synthesis of Compound 103

To a solution of ester 102 (15.3 g, 38.6 mmol, 1.0 eq.) in THF (100 mL)and methanol (100 mL) was added LiOH·H₂O (16.3 g, 389 mmol, 10.0 eq.) inwater (190 mL) at room temperature. The mixture was stirred at roomtemperature for 40 min. and then diluted with water (400 mL) and 1NKHSO₄ was added dropwise until pH 3-4 was reached. After extraction withEtOAc (3×300 mL), the organic phase was washed with brine, dried overanhydrous Na₂SO₄, filtered, concentrated to give 103 as a yellow solid(14.4 g, theoretical yield). ¹H NMR (500 MHz, CDCl₃) δ 10.48 (s, 1H),7.98-7.88 (m, 1H), 7.42 (dd, J=18.4, 8.2 Hz, 1H), 7.14-7.03 (m, 1H),4.48 (d, J=8.6 Hz, 1H), 3.90 (s, 1H), 2.82-2.53 (m, 3H), 1.97-1.82 (m,2H), 1.42-1.27 (m, 10H), 1.21 (d, J=6.7 Hz, 4H). MS ESI m/z calcd forC₁₇H₂₃N₂O₇ [M−H]⁻ 367.16, found 367.14.

Example 42. Synthesis of Compound 104

In a hydrogenation bottle, Pd/C (2.60 g, 10 wt %, 50% water) was addedto a solution of compound 103 (26.0 g, 70.6 mmol, 1.0 eq.) in methanol(260 mL). The mixture was shaken overnight under 1 atm H₂ then filteredthrough Celite (filter aid), the filtrate was concentrated to affordcompound 104 as a green oil (24.0 g, >100% yield).

Example 43. Synthesis of Compound 106

A mixture of tert-butyl-2-bromopropanoate (255 g, 1.22 mol, 1.0 eq.) andtriphenyl phosphine (320 g, 1.22 mol, 1.0 eq.) in dry acetonitrile (1 L)was stirred at room temperature for 18 h. Acetonitrile was removed underreduced pressure and toluene was added to crash out a white precipitate.Toluene was then decanted off and the white solid was dissolved indichloromethane (1 L) and transferred to a separatory funnel. 10% NaOH(1 L) was added to the funnel, and the organic layer immediately turnedyellow after shaking. The organic layer was separated and the aqueouslayer was extracted with dichloromethane (1 L) once. The dichloromethanelayers were combined and washed with brine (400 mL) once, then driedover Na₂SO₄, filtered and concentrated, giving the ylide 106 as a yellowsolid (280 g, 58%).

Example 44. Synthesis of Compound 107

Aldehyde 99 (450 g, 1.27 mol, 1.0 eq.) was dissolved in drydichloromethane (3 L), to which tert-butyl ester ylide 106 (546 g, 1.40mmol, 1.1 eq.) was added and the solution was stirred at r.t. overnightas determined complete by TLC. Purification by column chromatography(10-50% EtOAc/hexanes) afforded compound 107 (444 g, 75% yield) as awhite solid. ESI m/z calcd for C₂₈H₃₈NO₅ [M+H]⁺: 468.27, found 468.22.

Example 45. Synthesis of Compound 108

Compound 107 (63 g, 0.13 mol) was dissolved in methanol (315 mL) andhydrogenated (1 atm H₂) with Pd/C catalyst (10 wt %, 6.3 g) at r.t.overnight. The catalyst was filtered off and the filtrate wereconcentrated under reduced pressure to afford compound 108 (45.8 g, 93%yield).

Example 46. Synthesis of Compound 109

To a solution of compound 108 (390 g, 1.03 mol, 1.0 eq.) in THF (4 L)tert-butyl nitrite (1.06 kg, 10.3 mol, 10 eq.) was added at r.t. and thereaction was stirred overnight. After removal of THF, the residue waspurified by column chromatography (10-50% EtOAc/hexanes) to affordcompound 109 (314 g, 72% yield) as a light yellow solid.

Example 47. Synthesis of Compound 110

To a solution of 109 (166 g, 0.392 mol, 1.0 eq.) in EtOAc (500 mL) wasadded Pd/C (10 wt %, 16 g) under nitrogen, and the reaction flask wasevacuated and purged with hydrogen for 3 times. The reaction mixture wasstirred under hydrogen (1 atm) at r.t. for 16 h and then filtered overCelite and concentrated to afford product 110 (146 g, 97% yield) as alight yellow foam. ¹H NMR (400 MHz, CDCl₃) δ 6.62 (d, J=7.9 Hz, 1H),6.55 (s, 1H), 6.43 (d, J=7.3 Hz, 1H), 4.39 (dd, J=53.0, 44.2 Hz, 1H),3.77 (s, 4H), 2.72-2.29 (m, 3H), 1.83-1.58 (m, 1H), 1.40 (d, J=7.6 Hz,18H), 1.24 (s, 1H), 1.06 (t, J=5.7 Hz, 3H). MS ESI m/z calcd forC₂₁H₃₅N₂O₅ [M+H]⁺394.25, found 395.25.

Example 48. Synthesis of Compound 114

To a solution of (S)-4-isopropyloxazolidin-2-one (5.00 g, 38.7 mmol, 1.0eq.) in anhydrous THF (200 mL) at −78° C. was added n-BuLi (2.5 M inhexanes, 17.0 mL, 1.2 eq.) in 30 min under N₂. The mixture was stirredat −78° C. for 1 h, and then propionyl chloride (4.0 mL, 42.58 mmol, 1.1eq.) was added dropwise. After the mixture was stirred at −78° C. foranother 1 h, TLC analysis indicated the reaction completed. Saturatedammonium chloride solution (250 mL) was added and extracted with EtOAc(3×100 mL). The combined organic layers were washed with 1N NaOHsolution (200 mL) and brine (300 mL), dried over anhydrous Na₂SO₄,filtered, concentrated and purified by column chromatography (7:1hexanes/EtOAc) to give compound 114 as a colourless oil (6.36 g, 89%yield). MS ESI m/z calcd for C₉H₁₆NO₃ [M+H]⁺ 186.10, found 186.10. ¹HNMR (400 MHz, CDCl₃) δ 4.48-4.39 (m, 1H), 4.27 (t, J=8.7 Hz, 1H), 4.21(dd, J=9.1, 3.1 Hz, 1H), 3.06-2.82 (m, 2H), 2.38 (dtd, J=14.0, 7.0, 4.0Hz, 1H), 1.17 (t, J=7.4 Hz, 3H), 0.90 (dd, J=17.0, 7.0 Hz, 6H).

Example 49. Synthesis of Compound 115

To a solution of (S)-4-isopropyl-3-propionyloxazolidin-2-one (2.00 g,11.9 mmol, 1.1 eq.) in anhydrous dichloromethane (20 mL) at 0° C. wasadded DIPEA (2.3 mL, 12.9 mmol, 1.2 eq.) and n-Bu₂BOTf (1.0 M indichloromethane, 12.0 mL, 1.1 eq.) under N₂. The mixture was stirred at0° C. for 45 min, then cooled to −78° C., to which a solution ofcompound 99 (4.24 mL, 10.8 mmol, 1.0 eq.) in dichloromethane was addeddropwise. The mixture was stirred at −78° C. for 1 h and then warmedslowly to room temperature. The mixture was stirred at room temperatureovernight, and PBS (0.1M, pH 7.0, 100 mL) was added. After phaseseparation, the aqueous phase was further extracted with dichloromethane(3×50 mL). The combined organic layers were washed with brine (200 mL),dried over anhydrous Na₂SO₄, filtered and concentrated. The crudeproduct was re-dissolved in methanol (100 mL) and treated with H₂O₂ (30%aqueous solution, 26 mL, 23 eq.) at 0° C. for 3 h. The methanol wasremoved by rotary evaporation and water (100 mL) was added. Theresulting mixture was extracted with EtOAc (3×100 mL). The combinedorganic layers were washed with brine (300 mL), dried over anhydrousNa₂SO₄, filtered, concentrated and purified by SiO₂ columnchromatography (3:1 hexanes/EtOAc) to give compound 115 as a foamy solid(2.70 g, 49% yield). ¹H NMR (400 MHz, CDCl₃) δ7.52-7.26 (m, 5H), 7.15(d, J=7.4 Hz, 2H), 6.93 (d, J=7.3 Hz, 2H), 5.05 (s, 2H), 4.69 (d, J=7.0Hz, 1H), 4.47 (s, 1H), 4.36 (t, J=7.8 Hz, 1H), 4.17 (d, J=8.5 Hz, 1H),3.93 (d, J=7.1 Hz, 1H), 3.85 (s, 2H), 2.84 (d, J=6.9 Hz, 2H), 2.31 (s,1H), 1.40-1.37 (m, 9H), 1.31 (s, 3H), 0.92 (dd, J=13.4, 6.6 Hz, 6H). MSESI m/z calcd for C₃₀H₄₁N₂O₇ [M+H]+ 541.28, found 541.30.

Example 50. Synthesis of Compound 116

A mixture of compound 115 (2.50 g, 4.63 mmol, 1.0 eq.) and1,1′-thiocarbonyl-diimidazole (2.48 g, 13.89 mmol, 3.0 eq.) in anhydrousTHF (46 mL) was refluxed overnight. Water (100 mL) was added and theresulting mixture was extracted with EtOAc (3×50 mL). The combinedorganic layers were washed with brine (200 mL), dried over anhydrousNa₂SO₄, filtered, concentrated and purified by SiO₂ columnchromatography (3:1 hexanes/EtOAc) to give compound 116 as a yellow foam(2.33 g, 77% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.41 (s, 1H), 7.67 (s,1H), 7.36 (dt, J=16.0, 6.9 Hz, 6H), 7.09 (s, 1H), 7.05 (d, J=8.4 Hz,2H), 6.86 (d, J=8.4 Hz, 2H), 6.32 (d, J=9.5 Hz, 1H), 5.01 (s, 2H),4.56-4.43 (m, 2H), 4.32 (ddd, J=16.2, 15.6, 7.8 Hz, 3H), 4.19 (d, J=8.7Hz, 1H), 2.96 (dd, J=14.6, 4.4 Hz, 1H), 2.49 (dd, J=14.5, 10.5 Hz, 1H),2.29 (td, J=13.4, 6.7 Hz, 1H), 1.31 (s, 3H), 1.29 (s, 9H), 0.91 (dd,J=13.9, 6.9 Hz, 6H). MS ESI m/z calcd for C₃₄H₄₃N₄O₇S [M+H]⁺ 651.27,found 651.39.

Example 51. Synthesis of Compound 117

To a solution of compound 116 (1.90 g, 2.92 mmol, 1.0 eq.) in anhydroustoluene (30 mL) was added n-Bu₃SnH (1.6 mL, 5.84 mmol, 2.0 eq.) andazodiisobutyronitrile (0.05 g, 0.584 mmol, 0.1 eq.) in sequence. Themixture was refluxed for 2.5 h and then concentrated and purified bySiO₂ column chromatography (5:1 hexanes/EtOAc) to give compound 117 as awhite foam (1.21 g, 79% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.36 (ddd,J=24.5, 14.5, 7.1 Hz, 5H), 7.08 (d, J=8.5 Hz, 2H), 6.90 (d, J=8.5 Hz,2H), 5.04 (d, J=5.1 Hz, 2H), 4.48 (d, J=4.2 Hz, 1H), 4.33 (t, J=8.4 Hz,1H), 4.22 (d, J=9.7 Hz, 1H), 4.15 (d, J=8.8 Hz, 1H), 3.81 (s, 2H), 2.73(dd, J=14.1, 5.9 Hz, 1H), 2.61 (dd, J=14.0, 7.2 Hz, 1H), 2.29 (dq,J=13.5, 6.8 Hz, 1H), 2.11-2.00 (m, 1H), 1.35 (s, 9H), 1.20 (d, J=6.9 Hz,3H), 0.89 (dd, J=14.0, 6.9 Hz, 6H). MS ESI m/z calcd for C₃₀H₄₁N₂O₆[M+H]⁺ 525.28, found 525.37.

Example 52. Synthesis of Compound 118

To a solution of compound 117 (1.20 g, 2.29 mmol, 1.0 eq) in THF (30 mL)were added LiOH (0.192 g, 4.58 mmol, 2.0 eq.) in water (6 mL) and H₂O₂(30% aqueous solution, 1.4 mL, 6.0 eq.). After 3 h of stirring at 0° C.,sodium bisulfite solution (1.5 M, 30 mL) was added to quench thereaction. After 30 min, 1 N KHSO₄ was added dropwise until pH 4 wasreached. The reaction mixture was then extracted with EtOAc (3×50 mL).The EtOAc solution was washed with brine, dried over anhydrous Na₂SO₄,filtered, concentrated and purified by SiO₂ column chromatography (3:1hexanes/EtOAc, containing 1% HOAc) to give compound 118 as a white solid(0.78 g, 82% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.46-7.28 (m, 5H), 7.07(d, J=7.7 Hz, 2H), 6.91 (d, J=7.8 Hz, 2H), 4.52 (d, J=8.5 Hz, 1H), 3.87(d, J=41.8 Hz, 1H), 2.82-2.43 (m, 3H), 1.85 (t, J=12.2 Hz, 1H), 1.41 (s,9H), 1.17 (d, J=6.9 Hz, 3H). MS ESI m/z calcd for C₂₄H₃₂NO₅ [M+H]⁺414.22, found 414.21.

Example 53. Synthesis of Compound 119

A mixture of compound 118 (0.77 g, 1.86 mmol, 1.0 eq.) and Pd/C (10%0,0.25 g) in methanol (15 mL) was hydrogenated under 1 atm H₂ pressure for16 h and then filtered through Celite (filter aid). The filtrate wasconcentrated to afford compound 119 as a white solid (0.58 g, 96%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.00 (d, J=7.5 Hz, 2H), 6.80 (s, 2H),4.51 (d, J=9.0 Hz, 1H), 3.88 (s, 1H), 2.66 (dd, J=65.6, 22.6 Hz, 4H),1.88 (t, J=12.2 Hz, 1H), 1.42 (s, 9H), 1.14 (d, J=6.6 Hz, 3H). MS ESIm/z calcd for C₁₇H₂₆NO₅ [M+H]⁺: 324.17, found 324.16.

Example 54. Synthesis of Compound 120

To a solution of compound 119 (0.57 g, 1.76 mmol, 1.0 eq.) in THF (10mL) was added t-BuONO (0.63 mL, 5.28 mmol, 3.0 eq.) at 0° C. Thereaction was stirred at 0° C. for 1 hr then room temperature 1 h. Afterwater (50 mL) was added, the reaction mixture was extracted with EtOAc(3×30 mL). The combined organic layers were washed with brine (100 mL),dried over anhydrous Na₂SO₄, filtered, concentrated and purified by SiO₂column chromatography (2:1 hexanes/EtOAc, containing 1% HOAc) to givecompound 120 as a yellow solid (0.50 g, 77% yield). ¹H NMR (400 MHz,DMSO) δ7.92 (s, 1H), 7.47 (d, J=8.3 Hz, 1H), 7.05 (d, J=8.5 Hz, 1H),3.73 (s, 1H), 2.78 (dd, J=13.6, 5.3 Hz, 1H), 2.69-2.47 (m, 2H), 1.87 (t,J=11.9 Hz, 1H), 1.47-1.37 (m, 1H), 1.32 (s, 9H), 1.17 (d, J=7.2 Hz, 3H).MS ESI m/z calcd for C₁₇H₂₅N₂O₇ [M+H]⁺ 369.15, found 369.14.

Example 55. Synthesis of Compound 121

A mixture of compound 120 (0.50 g, 1.36 mmol, 1.0 eq.) and Pd/C (10 wt%, 0.02 g) in methanol (10 mL) was hydrogenated (1 atm H₂) at r.t. for 1h, and then filtered through Celite (filter aid). The filtrate wasconcentrated to afford compound 121 as white foam (0.43 g, 93% yield).MS ESI m/z calcd for C₁₇H₂₇N₂O₅ [M+H]+ 339.18, found 339.17. ¹H NMR (400MHz, MeOD) δ 6.60 (d, J=7.9 Hz, 2H), 6.44 (d, J=7.3 Hz, 1H), 3.71 (d,J=6.3 Hz, 1H), 2.62-2.37 (m, 3H), 1.83 (ddd, J=13.7, 9.9, 3.7 Hz, 1H),1.39 (s, 9H), 1.13 (d, J=7.1 Hz, 3H).

Example 56. Synthesis of Compound 124

To a solution of maleic anhydride (268 g, 2.73 mol) in acetic acid (1 L)was added 4-aminobutanoic acid (285 g, 2.76 mol). After stirring at r.t.for 30 min, the reaction was refluxed for 1.5 h, cooled to r.t. andevaporated under vacuum to give a residue, which was taken up in EA,washed with water and brine, and dried over anhydrous Na₂SO₄, filteredand concentrated. The crude product was crystallized from EtOAc and PEto give a white solid (400 g, 80% yield). ¹H NMR (500 MHz, CDCl₃) δ 6.71(s, 2H), 3.60 (t, J=6.7 Hz, 2H), 2.38 (t, J=7.3 Hz, 2H), 2.00-1.84 (m,2H).

Example 57. Synthesis of Compound 125

Compound 124 (400 g, 2.18 mol, 1.0 eq.) was dissolved in CH₂Cl₂ (1.5 L),to which N-hydroxysuccinimide (276 g, 2.40 mmol, 1.1 eq.) and DIC (303g, 2.40 mol, 1.1 eq.) were added at r.t. and stirred overnight. Thereaction was concentrated and purified by column chromatography (1:2petroleum ether/EtOAc) to give NHS ester 125 as a white solid (382 g,63% yield). ¹H NMR (500 MHz, CDCl₃) δ 6.74 (s, 2H), 3.67 (t, J=6.8 Hz,2H), 2.85 (s, 4H), 2.68 (t, J=7.5 Hz, 2H), 2.13-2.03 (m, 2H).

Example 58. Synthesis of Compound 126

To a solution of 124 (60 g, 328 mmol, 1.3 eq.) in THF (600 mL) was addedNMM (85.3 mL, 984 mmol, 3.0 eq.) at 0° C. with stirring, followed byisobutyl chloroformate (44.6 mL, 426 mmol, 1.3 eq.) dropwise. Afterstirring at 0° C. for 2 h, the resulting mixture was added dropwise to asolution of 104 (102 g, 259 mmol, 1.0 eq.) in THF (400 mL) while keepingthe temperature at 0° C. After the addition was completed, the reactionwas stirred for additional 30 min. and then quenched with water (300mL), extracted with EtOAc (3×300 mL). The combined organic layers weredried, filtered, concentrated and purified by column chromatography witha gradient of 9-35% EtOAc/PE to afford compound 126 (104 g, 73% yield)as a light yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.86 (s, 1H), 8.40(d, J=17.3 Hz, 1H), 6.87 (s, 3H), 6.70 (s, 2H), 4.53-4.16 (m, OH), 3.79(s, 1H), 3.62 (t, J=6.1 Hz, 1H), 2.63 (s, 1H), 2.40 (t, J=6.9 Hz, 1H),2.12-1.88 (m, 4H), 1.84-1.64 (m, 1H), 1.38 (t, J=9.6 Hz, 6H), 1.06 (t,J=6.0 Hz, 3H).

Example 59. Synthesis of Compound 127

Compound 126 (12.7 g, 22.7 mmol) dissolved in CH₂Cl₂ (20 mL) was treatedwith TFA (40 mL) at 0° C. and the reaction was warmed to r.t. andstirred for 3 h. The mixture was concentrated and co-evaporated withtoluene three times. The residue was triturated with diethyl ether and alight yellow solid 127 was collected (11.4 g, theoretical yield).

Example 60. Synthesis of Compound 128

To a solution of carboxylic acid 33 (40 mg, 0.074 mmol, 1.0 eq.) inEtOAc was added pentafluorophenol (27 mg, 0.148 mmol, 2.0 eq.) and DCC(23 mg, 0.111 mmol, 1.5 eq.). The reaction mixture was stirred at r.t.for 16 h and then filtered over a Celite pad, with washing of the padwith EtOAc. The filtrate was concentrated and re-dissolved in DMA (6mL), then compound 127 (56.6 mg, 0.13 mmol) and DIPEA (47.4 μL, 0.18mmol) were added. The reaction mixture was stirred at r.t. for 24 h andthen concentrated and purified by reverse phase HPLC (C₁₈ column,10-100% acetonitrile/water) to afford compound 128 (43 mg, 63% yield) asa white solid. MS ESI m/z calcd for C₄₆H₆₆N7O₁₁S [M+H]⁺ 924.45, found924.45.

Example 61. Synthesis of Compound 132

To a solution of compound 41a (11 g, 15.9 mmol, 1.0 eq.) and compound127 (12.3 g, 23.8 mmol, 1.5 eq.) in DMF (100 mL) was added DIPEA (6.9mL, 39.7 mmol, 2.5 eq.) at 0° C. The reaction mixture was warmed to r.t.and stirred for 1 h. The mixture was concentrated under vacuum andpurified on silica gel column (100% DCM to 10% MeOH/DCM) to givecompound 132 (10 g, 69% yield) as an amorphous solid. MS ESI m/z calcdfor C₄₅H₆₅N₇O₁₁S [M+H]⁺ 912.45, found 912.45.

Example 62. Synthesis of Compound 204

To a solution of (R)-4-isopropyloxazolidin-2-one (203) (25.0 g, 0.194mol, 1.0 eq) in anhydrous THF (1150 mL) was added n-BuLi (85.0 mL, 0.213mol, 1.1 eq) at −78° C. under N₂ and the mixture was stirred at the sametemperature for 1 h, a large number of white solids formed. Thenpropionyl chloride (20.0 mL, 0.232 mol, 1.2 eq) was added at −78° C. andthe mixture was stirred at the same temperature for 1 h. After theconsumption of (S)-4-isopropyloxazolidin-2-one monitored by TLC, thesolution was poured into saturated ammonium chloride solution (1.2 L)and the mixture was extracted with EA (700 mL, 350 mL×2). The organicextract was washed with 1.0 N NaOH solution (1.0 L) and brine (1.0 L),dried over anhydrous sodium sulfate, filtered, concentrated in vacuo andpurified by SiO₂ column chromatography (PE:EA=10:1) to give the titlecompound as a colorless oil (32.6 g, 90.8%). ESI m/z: calcd for C₉H₁₇NO₃[M+H]⁺: 186.1, found 186.1. ¹H NMR (400 MHz, CDCl₃) δ 4.48-4.37 (m, 1H),4.27 (t, J=8.7 Hz, 1H), 4.21 (dd, J=9.1, 3.1 Hz, 1H), 3.04-2.82 (m, 2H),2.45-2.30 (m, 1H), 1.17 (t, J=7.4 Hz, 3H), 0.90 (dd, J=17.1, 7.0 Hz,6H).

Example 63. Synthesis of Compound 205

To a solution of (R)-4-isopropyl-3-propionyloxazolidin-2-one (18.4 g,99.5 mmol, 1.1 eq) in anhydrous DCM (200 mL) were added Bu₂BOTf (1 Mdichloromethane solution, 100 mL, 100 mmol, 1.1 eq) and DIPEA (19 mL,108.6 mmol, 1.2 eq) at 0° C. under N₂, and the mixture was stirred atthe same temperature for 45 min. A solution of aldehyde 99 (32.2 g, 90.5mmol, 1.0 eq) in dichloromethane (320 mL) was added at −78° C. andstirred at the same temperature for 1 h, then the solution was allowedto slowly warm to room temperature for 15 hours. The mixture was pouredinto 700 mL of potassium phosphate buffer (pH 7.0) and extracted withethyl acetate. The organic extract was washed with brine, dried overanhydrous sodium sulfate, filtered, and then concentrated in vacuo. Theresidue was dissolved in methanol (730 mL) and cooled to 0° C., then 30%H₂O₂ aqueous solution (225 mL) was added slowly, and the mixture wasstirred at the same temperature for 3 hours. After addition of water(750 mL), the mixture was concentrated in vacuo to remove methanol. Theresulting aqueous solution was extracted with ethyl acetate (500 mL, 150mL×2), and the organic extract was washed with 5% sodium hydrogencarbonate solution and brine, dried over anhydrous sodium sulfate,filtered, concentrated in vacuo and purified by SiO₂ columnchromatography (PE:EA=3:1) to give the title compound as a white foam(31.7 g, 64.8%). ESI m/z: calcd for C₃₀H₄₁N₂O₇ [M+H]⁺: 541.3, found541.3. ¹H NMR (400 MHz, CDCl₃) δ 7.49-7.29 (m, 5H), 7.17 (t, J=10.7 Hz,2H), 6.93 (d, J=7.0 Hz, 2H), 5.06 (s, 2H), 4.28 (dd, J=44.4, 36.4 Hz,3H), 4.04-3.52 (m, 3H), 3.11-2.73 (m, 2H), 2.35 (s, 1H), 1.41 (t, J=16.3Hz, 9H), 0.91 (dd, J=15.6, 6.4 Hz, 5H).

Example 64. Synthesis of Compound 206

To a solution of compound 205 (28.3 g, 52.3 mmol, 1.0 eq) in anhydrousTHF (350 mL) was added 1,1-thiocarbonyl diimidazole (TCDI) (35.1 g,157.0 mmol, 3.0 eq), and the mixture was heated under reflux overnight.After the consumption of starting material monitored by TLC, the mixturewas concentrated in vacuo and purified by SiO₂ column chromatography(PE:EA=3:1) to give the title compound as a pale yellow foam (26.1 g,76.8%). ESI m/z: calcd for C₃₄H₄₃N₄O₇S [M+H]⁺: 651.3, found 651.3. ¹HNMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.43 (d, J=11.8 Hz, 1H), 7.42-7.28(m, 5H), 7.06 (d, J=8.3 Hz, 2H), 7.01 (s, 1H), 6.80 (d, J=8.3 Hz, 2H),6.17 (dd, J=8.5, 2.9 Hz, 1H), 4.96 (s, 2H), 4.42-4.04 (m, 5H), 2.83 (dd,J=14.2, 6.2 Hz, 1H), 2.69 (dd, J=14.2, 7.1 Hz, 1H), 2.32 (dd, J=6.8, 4.2Hz, 1H), 1.37 (s, 9H), 1.30 (d, J=6.9 Hz, 3H), 0.87 (dd, J=9.9, 7.0 Hz,6H).

Example 65. Synthesis of Compound 207

To a solution of compound 206 (26.0 g, 40.0 mmol, 1.0 eq) in anhydroustoluene (350 mL) was added n-Bu₃SnH (21.5 mL, 80.0 mmol, 2.0 eq) and2,2′-azobis(2-methylpropionitrile) (AIBN) (0.066 g, 0.01 eq) under N₂,and the mixture was heated under reflux for 1 hour. After theconsumption of starting material monitored by TLC, the mixture wasconcentrated in vacuo and purified by SiO₂ column chromatography(PE:EA=5:1) to give the title compound as a white foam (6.0 g, 37.3%).ESI m/z: calcd for C₃₀H₄₁N₂O₆ [M+H]⁺: 525.3, found 525.3. ¹H NMR (400MHz, CDCl₃) δ 7.37 (ddd, J=25.1, 15.1, 7.1 Hz, 5H), 7.08 (d, J=7.9 Hz,2H), 6.89 (d, J=8.4 Hz, 2H), 5.03 (s, 2H), 4.61 (d, J=8.4 Hz, 1H), 4.40(s, 1H), 4.32-4.08 (m, 2H), 3.91-3.66 (m, 2H), 2.83 (d, J=8.4 Hz, 1H),2.60 (t, J=10.1 Hz, 1H), 2.33 (s, 1H), 1.71 (s, 1H), 1.41 (s, 9H), 1.15(d, J=6.5 Hz, 3H), 0.87 (dd, J=17.0, 7.0 Hz, 6H).

Example 66. Synthesis of Compound 208

To a solution of compound 207 (7.84 g, 15.0 mmol, 1.0 eq) in THF (90 mL)and water (30 mL) was added LiOH·H₂O (1.57 g, 37.5 mmol, 2.5 eq) in 30%H₂O₂ aqueous solution (11.4 mL, 112.5 mmol, 7.5 eq) at 0° C., and themixture was stirred at the same temperature for 3 hours. After additionof 1.5M Na₂SO₃ solution (160 mL) at 0° C., the mixture was stirred atthe same temperature for 30 min. then 1N KHSO₄ was added slowly until pH4. The resulting aqueous solution was extracted with EA (200 mL, 75mL×2), and the organic extract was washed with brine, dried overanhydrous sodium sulfate, filtered, concentrated in vacuo and purifiedby SiO₂ column chromatography (PE:EA=2:1) to give the title compound asa white solid (6.18 g, 100%). ESI m/z: calcd for C₂₄H₃₂N₁O₅ [M+H]⁺:414.2, found 414.2. ¹H NMR (400 MHz, CDCl₃) δ 7.39 (ddd, J=24.5, 15.0,7.2 Hz, 5H), 7.11 (d, J=7.8 Hz, 2H), 6.93 (d, J=8.3 Hz, 2H), 5.06 (s,2H), 4.44 (t, J=8.3 Hz, 1H), 3.83 (d, J=69.4 Hz, 1H), 2.85-2.61 (m, 2H),2.61-2.40 (m, 1H), 1.99-1.70 (m, 1H), 1.39 (d, J=26.1 Hz, 9H), 1.19 (s,3H).

Example 67. Synthesis of Compound 209

To a solution of compound 208 (6.18 g, 15.0 mmol, 1.0 eq) in MeOH (50mL) was added Pd/C (0.6 g, 10% Pd/C) in a hydrogenation bottle. Themixture was shaken under 1 atm hydrogen atmosphere overnight, thenfiltered. The filtrate was concentrated to give the title compound ascolourless oil (4.8 g, 99% yield). ESI m/z: calcd for C₁₇H₂₆N₁O₅ [M+H]⁺:324.2, found 324.2. ¹H NMR (400 MHz, CDCl₃) δ 6.97 (d, J=6.5 Hz, 2H),6.74 (d, J=8.2 Hz, 2H), 3.93-3.66 (m, 1H), 2.58 (tdd, J=19.5, 12.9, 7.4Hz, 3H), 1.75 (ddd, J=20.1, 16.3, 7.7 Hz, 1H), 1.37 (d, J=21.5 Hz, 9H),1.11 (d, J=7.0 Hz, 3H).

Example 68. Synthesis of Compound 210

To a solution of compound 209 (4.8 g, 15.0 mmol, 1.0 eq) in anhydrousTHF (75 mL) was added slowly t-BuONO (18.0 mL, 150 mmol, 10.0 eq) at 0°C. under N₂, and the mixture was stirred at the same temperature for 3hours. After the consumption of starting material monitored by TLC, 1NKHSO₄ was added slowly to the mixture until pH 4. The resulting aqueoussolution was extracted with EA (150 mL, 75 mL×2), and the organicextract was washed with brine, dried over anhydrous sodium sulfate,filtered, concentrated in vacuo and the residue was purified by SiO₂column chromatography (PE:EA=3:1) to give the title compound as a yellowsolid (3.6 g, 65.4%). ESI m/z: calcd for C₁₇H₂₅N₂O₇ [M+H]⁺: 369.2, found369.2. ¹H NMR (400 MHz, MeOD) δ 7.93 (d, J=2.0 Hz, 1H), 7.48 (dd, J=8.6,2.1 Hz, 1H), 7.06 (d, J=8.5 Hz, 1H), 3.83-3.71 (m, 1H), 2.82 (dd,J=13.6, 5.0 Hz, 1H), 2.66-2.41 (m, 2H), 1.84 (ddd, J=14.0, 10.6, 5.6 Hz,1H), 1.65-1.51 (m, 1H), 1.28 (d, J=24.9 Hz, 9H), 1.15 (d, J=7.0 Hz, 3H).

Example 69. Synthesis of Compound 211

To a solution of compound 210 (3.2 g, 7.74 mmol, 1.0 eq) in MeOH (20 mL)was added Pd/C (0.2 g, 10% Pd/C) in a hydrogenation bottle. The mixturewas shaken under 1 atm H₂ atmosphere for 3 h. After consumption ofstarting material monitored by TLC, the mixture was filtered and thefiltrate was concentrated to give the title compound as white foam (2.3g, 92.0% yield). ESI m/z: calcd for C₁₇H₂₇N₂O₅ [M+H]⁺: 339.2, found339.2. ¹H NMR (400 MHz, MeOD) δ 6.61 (d, J=8.0 Hz, 2H), 6.45 (d, J=6.3Hz, 1H), 3.72 (d, J=7.3 Hz, 1H), 2.68-2.34 (m, 3H), 1.81-1.66 (m, 1H),1.56-1.45 (m, 1H), 1.36 (d, J=29.0 Hz, 9H), 1.08 (d, J=6.9 Hz, 3H).

Example 70. Synthesis of Compound 390

To a solution of compound 102 (1.00 g, 2.52 mmol) in acetonitrile (10mL) was added CCl₄ (2.2 mL, 22.7 mmol, 9.0 eq.) at −25 OC. Afterstirring for 10 min, diisopropylethylamine (0.88 mL, 5.04 mmol, 2.0 eq.)and DMAP (0.03 g, 0.252 mmol, 0.1 eq.) were added, followed by dibenzylphosphite (0.84 mL, 3.78 mmol, 1.5 eq.). The reaction mixture wasallowed to reach r.t. over 1.5 h, and then quenched by a solution ofKH₂PO₄ (0.5 M, 50 mL). The reaction mixture was extracted with EtOAc(3×50 mL). The combined organic extracts were dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The residue was purifiedby flash column chromatography (10-50% EtOAc/PE) to afford compound 390(1.60 g, 96% yield) as a colorless oil. MS ESI m/z calcd forC₃₃H₄₁N₂O₁₀P [M+H]⁺ 657, found 657.

Example 71. Synthesis of Compound 391

To a solution of compound 390 (1.60 g, 2.43 mmol) in methanol (20 mL)was added Pd/C (10 wt %, 160 mg). The reaction mixture was stirred underH₂ atmosphere (1 atm) at r.t. for 3 h, then filtered through Celite andconcentrated under reduced pressure to afford compound 391 (1.00 g, 91%yield) as a white solid. MS ESI m/z calcd for C₁₉H₃₁N₂O₈P [M−H]⁻ 447,found 447.

Example 72. Synthesis of Compound 392

A solution of compound 391 (730 mg, 1.63 mmol) in ethanol (10 mL) wastreated with 1 N NaOH (16 mL, 16.3 mmol, 10 eq.) at r.t. overnight, andthen concentrated under reduced pressure. The residue was taken up inwater (20 mL) and acidified to pH 6 by 1 N HCl. The aqueous solution wasconcentrated under reduced pressure and the residue was triturated withMeOH/EtOAc (80:20, 5 mL), compound 392 (0.68 g, 990% yield) wascollected from filtration as a white solid. MS ESI m/z calcd forC₁₇H₂₇N₂O₈P [M−H]⁻ 417, found 417.

Example 73. Synthesis of Compound 399

2-(2-aminoethoxy)ethanol (21.00 g, 200 mmol, 1.0 eq.) and K₂CO₃ (83.00g, 600 mmol, 3.0 eq.) in acetonitrile (350 mL) was added BnBr (57.0 mL,480 mmol, 2.4 eq.). The mixture was refluxed overnight. Water (1 L) wasadded and extracted with EtOAc (3×300 mL). The combined organic layerswere washed with brine (1000 mL), dried over anhydrous Na₂SO₄, filtered,concentrated and purified by SiO₂ column chromatography (4:1hexanes/EtOAc) to give a colourless oil (50.97 g, 89.2% yield). MS ESIm/z calcd for C₁₈H₂₃NO₂Na [M+Na]⁺ 309.17, found 309.19.

Example 74. Synthesis of Compound 400

To a mixture of 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mmol,1.0 eq.), tert-butyl acrylate (72.0 mL, 495.9 mmol, 3.0 eq.) and n-Bu₄NI(6.10 g, 16.53 mmol, 0.1 eq.) in DCM (560 mL) was added sodium hydroxidesolution (300 mL, 50%). The mixture was stirred overnight. The organiclayer was separated and the water layer was extracted with EtOAc (3×100mL). The organic layers were washed with water (3×3000 mL) and brine(3000 mL), dried over anhydrous Na₂SO₄, filtered, concentrated andpurified by SiO₂ column chromatography (7:1 hexanes/EtOAc) to give acolourless oil (61.08 g, 89.4% yield). MS ESI m/z calcd for C₂₅H₃₆NO₄[M+H]⁺ 414.2566, found 414.2384.

Example 75. Synthesis of Compound 401

To a solution of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoate (20.00 g, 48.36 mmol, 1.0 eq.) in THF (30 mL) and MeOH (60mL) was added Pd/C (2.00 g, 10 wt %, 50% wet) in a hydrogenation bottle.The mixture was shaken overnight, filtered through Celite (filter aid),and the filtrate was concentrated to afford a colourless oil (10.58 g,93.8% yield). MS ESI m/z calcd for C₁₁H₂₄NO₄ [M+H]⁺ 234.1627, found234.1810.

Example 76. Synthesis of Compound 402

To a solution of (E)-3-bromoacrylic acid (0.15 g, 1 mmol), DMAP (0.15 g,1.2 mmol) and DCC (0.21 g, 1 mmol) in DCM (10 ml), compound 401 (0.23 g,1 mmol) were added at 0° C. The reaction mixture was allowed to warm tor.t. and stirred overnight. The crude product was concentrated andpurified by SiO₂ column chromatography with a gradient of EA/DCM to givethe title product 402 (0.31 g, 85% yield). ESI MS m/z: calcd forC₁₄H₂₅BrNO₅ [M+H]⁺: 366.08, found 366.08.

Example 77. Synthesis of Compound 403

Compound 402 (0.31 g, 0.84 mmol) was dissolved in fomic acid (4 mL) at0° C. then H₂O (2 mL) was added. The reaction mixture was allowed towarm to r.t. and stirred overnight. The crude product was concentratedand used for the next step without further purification. ESI MS m/z:calcd for C₁₀H₁₇BrNO₅ [M+H]⁺: 310.02, found 310.03.

Example 78. Synthesis of Compound 404

Compound 303 (0.12 g, 0.39 mmol), NHS (0.067 g, 0.58 mmol) and EDCI(0.11 g, 0.58 mmol) were dissolved in DCM (10 mL) and the mixture wasstirred at r.t. overnight, concentrated and purified by SiO₂ columnchromatography to give the title product 404 (0.13 g, 82% yield). ESI MSm/z: calcd for C₁₄H₂₀BrN₂O₇ [M+H]⁺: 407.04, found 407.04.

Example 79. Synthesis of Compound 426

A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150mmol) in H₂O (40 mL) was cooled to 0° C. and treated with a solution ofCbzCl (16.1 g, 95 mmol) in THF (32 ml) dropwise. After 1 h, the reactionwas allowed to warm to r.t. and stirred for 3 h. THF was removed undervacuum, the pH of the aqueous solution was adjusted to 1.5 by additionof 6 N HCl. The solution was extracted with ethyl acetate, and theorganic layer was washed with brine, dried and concentrated to givecompound 426 (16.4 g, 92% yield). MS ESI m/z calcd for C₁₂H₁₆NO₅ [M+H]⁺238.10, found 238.08.

Example 80. Synthesis of Compound 427

DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were added to asolution of 4-(((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2mmol) and t-BuOH (15.4 g, 208 mmol) in DCM (100 mL). After stirring atr.t. overnight, the reaction was filtered and filtrate concentrated. Theresidue was dissolved in ethyl acetate and the washed with 1N HCl, brineand dried over Na₂SO₄. Concentration and purification by columnchromatography (10 to 50% EtOAc/hexanes) yielded compound 427 (7.5 g,37% yield). MS ESI m/z calcd for C₁₆H₂₃NO₄Na [M+Na]⁺ 316.16, found316.13.

Example 81. Synthesis of Compound 428

Tert-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol)was dissolved in MeOH (50 mL), and mixed with Pd/C catalyst (10 wt %,100 mg) then hydrogenated (1 atm) at r.t. for 3 h. The catalyst wasfiltered off and all volatiles were removed under vacuum to affordcompound 428 (272 mg, 90% yield). MS ESI m/z calcd for C₈H₁₈NO₂ [M+H]⁺160.13, found 160.13.

Example 82. Synthesis of Compound 430

Tert-Butyl 4-aminobutanoate (477 mg, 3 mmol) and 2,3-dibromosuccinicacid (414 mg, 1.5 mmol) was dissolved in DCM (35 mL), to which DIPEA(1.16 g, 9 mmol) and EDC (0.86 g, 4.5 mmol) were added. The resultingsolution was stirred at r.t. overnight and then washed with brine, driedover Na₂SO₄. Filtration, concentration and purification by columnchromatography (pure DCM to 10% MeOH/DCM) yielded compound 430 (160 mg,22% yield). MS ESI m/z calcd for C₂₀H₃₄BrN₂O₆ [M+H]+ 477.15, found477.16.

Example 83. Synthesis of Compound 431

Compound 430 (80 mg, 0.168 mmol) was dissolved in DCM (5 mL) and treatedwith formic acid (8 mL) at 38° C. overnight. All volatiles were removedunder vacuum to afford compound 431 (61 mg, 99% yield). MS ESI m/z calcdfor C₁₂H₁₈BrN₂O₆ [M+H]⁺ 365.03, found 365.05.

Example 84. Synthesis of Compound 432

NHS (60 mg, 0.504 mmol) and EDCI (97 mg, 0.504 mmol) were added to asolution of compound 431 (61 mg, 0.168 mmol) in DCM (10 mL). Afterstirring at r.t. overnight, the reaction mixture was concentrated andpurified by column chromatography (0 to 10% MeOH/DCM) to afford compound432 (72 mg, 77% yield). MS ESI m/z calcd for C₂₀H₂₄BrN₄O₁₀ [M+H]⁺559.06, found 559.78.

Example 85. Synthesis of Compound 433

NaH₂PO₄ (0.1M in water, 1 mL) was added to a solution of compound 432(36 mg, 0.065 mmol) and compound 110 (25 mg, 0.063 mmol) in EtOH (5 mL).The resulting solution was stirred at r.t. overnight. All volatiles wereremoved under vacuum and the residue was purified by columnchromatography (0 to 10% MeOH/DCM) to yield compound 433 (19.7 mg, 41%yield). MS ESI m/z 741.35 ([M+H]⁺).

Example 86. Synthesis of Compound 435

Compound 433 (18 mg, 0.024 mmol) was dissolved in DCM (2 mL) and treatedwith TFA (2 mL) at r.t. for 2 h. All volatiles were removed under vacuumto afford compound 435 (14 mg, 98% yield), which was use directly in thenext step. MS ESI m/z 585.22 ([M+H]⁺).

Example 87. Synthesis of Compound 437

Compound 435 (14 mg, 0.0239 mmol) and perfluorophenyl ester 33a (18 mg,0.0255 mmol) were dissolved in DMA (5 mL). To the mixture, DIPEA (10 mg,0.077 mmol) was added. The resulting mixture was stirred at r.t.overnight, concentrated and purified by preparative HPLC (C₁₈ column,10-90% MeCN/H₂O) to afford compound 437 (12.8 mg, 48% yield). MS ESI m/z1105.50 ([M+H]⁺).

Example 88. Synthesis of Compound 441

To a solution of 2,2′-(ethane-1,2-diylbis(oxy))diethanol (55.0 mL,410.75 mmol, 3.0 eq.) in anhydrous THF (200 mL) was added sodium (0.1g). The mixture was stirred until Na disappeared and then tert-butylacrylate (20.0 mL, 137.79 mmol, 1.0 eq.) was added dropwise. The mixturewas stirred overnight and then quenched by HCl solution (20.0 mL, 1N) at0° C. THF was removed by rotary evaporation, brine (300 mL) was addedand the resulting mixture was extracted with EtOAc (3×100 mL). Theorganic layers were washed with brine (3×300 mL), dried over anhydrousNa₂SO₄, filtered and concentrated to afford a colourless oil (30.20 g,79.0% yield), which was used without further purification. MS ESI m/zcalcd for C₁₃H₂₇O₆ [M+H]⁺ 278.1729, found 278.1730.

Example 89. Synthesis of Compound 442

To a solution of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (30.20 g, 108.5 mmol, 1.0 eq.) and TsCl (41.37 g, 217.0 mmol,2.0 eq.) in anhydrous DCM (220 mL) at 0° C. was added TEA (30.0 mL,217.0 mmol, 2.0 eq.). The mixture was stirred at room temperatureovernight, and then washed with water (3×300 mL) and brine (300 mL),dried over anhydrous Na₂SO₄, filtered, concentrated and purified by SiO₂column chromatography (3:1 hexanes/EtOAc) to give a colourless oil (39.4g, 84.0% yield). MS ESI m/z calcd for C₂₀H₃₃O₈S [M+H]⁺ 433.1818, found433.2838.

Example 90. Synthesis of Compound 443

To a solution of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (39.4 g, 91.1 mmol, 1.0 eq.) in anhydrous DMF (100 mL) wasadded NaN₃ (20.67 g, 316.6 mmol, 3.5 eq.). The mixture was stirred atroom temperature overnight. Water (500 mL) was added and extracted withEtOAc (3×300 mL). The combined organic layers were washed with water(3×900 mL) and brine (900 mL), dried over anhydrous Na₂SO₄, filtered,concentrated and purified by SiO₂ column chromatography (5:1hexanes/EtOAc) to give a light yellow oil (23.8 g, 85.53% yield). MS ESIm/z calcd for C₁₃H₂₅O₃N₅Na [M+Na]⁺ 326.2, found 326.2.

Example 91. Synthesis of Compound 444

Raney-Ni (7.5 g, suspended in water) was washed with water (three times)and isopropyl alcohol (three times) and mixed with compound 443 (5.0 g,16.5 mmol) in isopropyl alcohol. The mixture was stirred under a H₂balloon at r.t. for 16 h and then filtered over a Celite pad, withwashing of the pad with isopropyl alcohol. The filtrate was concentratedand purified by column chromatography (5-25% MeOH/DCM) to give lightyellow oil (2.60 g, 57% yield). MS ESI m/z calcd for C₁₃H₂₈NO₅ [M+H]⁺279.19; found 279.19.

Example 92. Synthesis of Compound 445

Acetylenedicarboxylic acid (0.35 g, 3.09 mmol, 1.0 eq.) was dissolved inNMP (10 mL) and cooled to 0° C., to which compound 444 (2.06 g, 7.43mmol, 2.4 eq.) was added, followed by DMTMM (2.39 g, 8.65 mmol, 2.8 eq.)in portions. The reaction was stirred at 0° C. for 6 h and then dilutedwith ethyl acetate and washed with water and brine. The organic solutionwas concentrated and triturated with a mixture solvent of ethyl acetateand petroleum ether. The solid was filtered off and the filtrate wasconcentrated and purified by column chromatography (80-90% EA/PE) togive a light yellow oil (2.26 g, >100% yield), which was used withoutfurther purification. MS ESI m/z calcd for C₃₀H₅₃N₂O₁₂ [M+H]⁺ 633.35;found 633.30.

Example 93. Synthesis of Compound 446

Compound 445 (2.26 g) was dissolved in dichloromethane (15 mL) andcooled to 0° C. then treated with TFA (15 mL). The reaction was warmedto r.t. and stirred for 45 min, and then the solvent and residual TFAwas removed on rotovap. The crude product was purified by columnchromatography (0-15% MeOH/DCM) to give light yellow oil (1.39 g, 86%yield for two steps). MS ESI m/z calcd for C₂₂H₃₇N₂O₁₂ [M+H]⁺ 521.23;found 521.24.

Example 94. Synthesis of Compound 480

Compound 110 (68 mg, 0.17 mmol), compound 124 (94.5 mg, 0.52 mmol) andHATU (162 mg, 0.425 mmol) were dissolved in DCM (50 mL). TEA (73 ul,0.52 mmol) was then added. The reaction mixture was stirred at r.t.overnight. Then the solvent was removed under reduced pressure and theresidue was purified by SiO₂ column to give the title product 480 (98mg, 80% yield). ESI m/z calcd for C₃₇H₄₉N₄O₁₁ [M+H]⁺: 725.33, found725.34.

Example 95. Synthesis of Compound 481

Compound 480 (98 mg, 0.135 mmol) dissolved in DCM (1.0 mL) was treatedwith TFA (1.0 mL) at r.t. for 2 h, then concentrated and redissolved inDMA (1 mL), to which pentafluorophenyl ester 41a (44 mg, 0.06 mmol) andDIPEA (45.8 μL, 0.27 mmol) were added. The reaction was stirredovernight and then concentrated. The residue was purified by prep-HPLCwith a gradient of MeCN/H₂O to give the title product 481 (37 mg, 55%yield). ESI m/z calcd for C₅₃H₇₃N₈O₁₄S [M+H]⁺: 1077.49, found 1077.50.

Example 96. Synthesis of Compound 484

To a solution of (S)-2-amino-3-(4-nitrophenyl)propanoic acid (13.2 g,62.8 mmol) in methanol (120 mL) was added thionyl chloride (9 mL, 125.6mmol) at 0° C. The reaction mixture was heated to reflux and stirred for1 h, then concentrated under vacuum and suspended in ethyl acetate (50mL). The mixture was then filtered to afford the title compound as awhite solid (14.5 g, 91% yield). ESI m/z calcd for C₁₀H₁₃N₂O₄ [M+H]⁺:225.08, found 225.08.

Example 97. Synthesis of Compound 485

To a solution of compound 484 (9.5 g, 36.4 mmol) in THF (200 mL) wasadded triethylamine (12.6 mL, 91.1 mmol). After the mixture was stirredfor 30 minutes, di-tert-butyl dicarbonate (12.5 mL, 54.7 mmol) wasadded, and the reaction mixture was stirred for 1 h, then diluted withethyl acetate (200 mL), washed with 1 N HCl (30 mL), water (30 mL),dried over sodium sulfate, filtered and concentrated under vacuum toafford the title compound as a white solid (11.4 g, 97% yield). ESI m/zcalcd for C₁₅H₂₁N₂O₆ [M+H]⁺: 325.13, found 325.13.

Example 98. Synthesis of Compound 486

To a solution of compound 485 (14 g, 43.2 mmol) in anhydrousdichloromethane (150 mL) was added DIBAL-H (108 mL, 108 mmol) at −78° C.The reaction mixture was stirred at −78° C. for 30 min., then pouredinto ice water (200 mL), extracted with ethyl acetate (3×80 mL). Thecombined organic phase was washed with 1N HCl (2×50 mL), water (50 mL),dried over sodium sulfate, filtered, concentrated under vacuum, andpurified by silica gel column chromatography to afford the titlecompound (8.6 g, 68% yield). ESI m/z calcd for C₁₄H₁₉N₂O₅ [M+H]⁺:295.12, found 295.12.

Example 99. Synthesis of Compound 487

To a solution of compound 106 (8.1 g, 20.8 mmol) in DCM (100 mL) wasadded compound 486 (5.2 g, 17.8 mmol) at 0° C. The reaction mixture waswarmed to r.t. and stirred for 30 min. then concentrated under vacuumand purified by silica gel column to afford the title compound as ayellow solid (5.9 g, 82% yield). ESI m/z calcd for C₂₁H₃₁N₂O₆ [M+H]⁺:406.21, found 406.21.

Example 100. Synthesis of Compound 488

To a solution of compound 487 (4 g, 9.85 mmol) in MeOH (40 mL) was addedPd/C (0.4 g, 10 wt %) in a hydrogenation bottle. The mixture was stirredunder 1 atm H₂ overnight, filtered through Celite (filter aid), and thefiltrate was concentrated to afford compound 488 (3.6 g, yield-100%).ESI m/z: calcd for C₂₁H₃₅N₂O₄ [M+H]⁺: 379.25, found 379.25.

Example 101. Synthesis of Compound 489

To a solution of compound 488 (3.6 g, 9.52 mmol) and triethylamine (1.3mL, 9.52 mmol) in dichloromethane (50 mL) was added4-nitrobenzenesulfonyl chloride (2.1 g, 9.52 mmol) at 0° C. The reactionmixture was warmed to r.t. and stirred for 1 h, then diluted with DCM(50 mL), washed with 1N HCl (20 mL), water (20 mL), dried over sodiumsulfate, filtered and concentrated under vacuum, then purified by silicagel column chromatography to afford the title compound as a yellow solid(4 g, 75% yield). ESI m/z calcd for C₂₇H₃₈N₃O₈S [M+H]⁺: 564.23, found564.23.

Example 102. Synthesis of Compound 490

To a solution of compound 489 (3.6 g, 6.39 mmol) in acetonitrile (40 mL)was added tert-butyl nitrite (2.29 mL, 19.1 mmol). The reaction mixturewas warmed to 45° C. and stirred for 6 hours. The reaction was thenconcentrated under vacuum and purified by silica gel columnchromatography to afford the title compound (3 g, 79% yield). ESI m/zcalcd for C₂₇H₃₇N₄O₁₀S [M+H]⁺: 609.22, found 609.22.

Example 103. Synthesis of Compound 491

To a solution of compound 490 (3.0 g, 4.92 mmol) in acetonitrile/DMSO(30 mL/1 mL) were added 4-methoxy thiophenol (2.76 g, 19.7 mmol) andpotassium carbonate (2.7 g, 19.7 mmol). The reaction mixture was stirredat the room temperature overnight, then diluted with ethyl acetate (100mL), washed with water (20 mL), brine (20 mL), dried over sodiumsulfate, filtered and concentrated under vacuum, and purified by silicagel column chromatography to afford the title compound (1.7 g, 85%yield). ESI m/z calcd for C₂₁H₃₄N₃O₆ [M+H]⁺: 424.24, found 424.24.

Example 104. Synthesis of Compound 492

To a solution of compound 491 (100 mg, 0.236 mmol) in MeOH (4 mL) wasadded Pd/C (10 mg, 10 wt %) in a hydrogenation bottle. The mixture wasstirred under 1 atm H₂ overnight, filtered through Celite (filter aid),and the filtrate was concentrated to afford the title compound (92.9 mg,—100% yield). ESI m/z calcd for C₂₁H₃₆N₃O₄ [M+H]⁺: 394.26, found 394.26.

Example 105. Synthesis of Compound 493

Compound 492 (66 mg, 0.17 mmol), compound 124 (94.5 mg, 0.52 mmol) andHATU (162 mg, 0.425 mmol) were dissolved in DCM (50 mL). TEA (73 ul,0.52 mmol) was then added. The reaction mixture was stirred at r.t.overnight, the solvent was removed under reduced pressure and theresidue was purified by SiO₂ column to give the title product 493 (98mg, 80% yield). ESI m/z calcd for C₃₇H₅₀N₅O₁₀ [M+H]⁺: 724.35, found724.35.

Example 106. Synthesis of Compound 494

Compound 493 (98 mg, 0.135 mmol) dissolved in DCM (1.0 mL) was treatedwith TFA (1.0 mL) at r.t. for 2 h, then concentrated to give compound494, which was used in the next step without further purification.

Example 107. Synthesis of Compound 495

To a solution of compound 494 (76.9 mg, 0.135 mmol) in DMA (1 mL) wasadded pentafluorophenyl ester 41a (44 mg, 0.06 mmol) and DIPEA (45.8 μL,0.27 mmol). The reaction was stirred overnight, then concentrated andthe residue was purified by prep-HPLC with a gradient of MeCN/H₂O togive the title product 495 (37 mg, 55% yield). ESI m/z calcd forC₅₃H₇₄N₉O₁₃S [M+H]⁺: 1076.50, found 1076.50.

Example 108. Synthesis of Compound 509

To a solution of maleimide (6.35 g, 65.4 mmol, 1.0 eq.) in EtOAc (120mL) were added N-methyl morpholine (8.6 mL, 78.5 mmol, 1.2 eq.) andmethyl chloroformate (6.0 mL, 78.5 mmol, 1.2 eq.) at 0° C. The reactionwas stirred at 0° C. for 30 min and r.t. 1 h. The solid was filtered offand filtrate concentrated. The residue was dissolved in CH₂Cl₂ andfiltered through a silica gel plug and eluted with CH₂Cl₂ to remove thecolor. The appropriate fractions were concentrated and resulted solidwas triturated with 10% EtOAc/PE to give a white solid (9.00 g, 89%yield).

Example 109. Synthesis of Compound 510

A mixture of compound 401 (8.16 g, 35.0 mmol, 1.0 eq.) and saturatedNaHCO₃ (40 mL) was cooled to 0° C., to which compound 509 (5.43 g, 35.0mmol, 1.0 eq.) was added in portions. After stirring at 0° C. for 1 h,the reaction was warmed to r.t. and stirred for 1 h. The reaction wasextracted with DCM (3×100 mL) and the organic extract was washed withbrine, dried over anhydrous Na₂SO₄, concentrated and purified by SiO₂column chromatography to give a white solid (6.76 g, 62% yield). MS ESIm/z calcd for C₁₅H₂₃NO₆ [M+H]⁺ 314.15, found 314.15.

Example 110. Synthesis of Compound 511

A solution of compound 510 (1.85 g, 5.9 mmol) was dissolved in DCM (20mL) and treated with TFA (7 mL) at r.t. for 16 h, then concentrated andpurified by SiO₂ column chromatography (11:1 DCM/MeOH) to give a whitefoam (1.47 g, 97% yield). MS ESI m/z calcd for C₁₁H₁₅NO₆ [M+H]⁺ 258.09,found 258.09.

Example 111. Synthesis of Compound 519

A mixture of N-Boc-ethylenediamine (5.6 mL, 35.4 mmol, 1.1 eq.) andsaturated NaHCO₃ (60 mL) was cooled to 0° C., to which compound 509(5.00 g, 32.2 mmol, 1.0 eq.) was added in portions. After stirring at 0°C. for 30 min, the reaction was warmed to r.t. and stirred for 1 h. Theprecipitate was collected by filtration and washed with cold water, thendissolved in EtOAc and washed with brine, dried over anhydrous Na₂SO₄and concentrated to give a white solid (6.69 g, 87% yield).

Example 112. Synthesis of Compound 520

A solution of compound 519 (6.00 g, 25.0 mmol), furan (18.0 mL) intoluene (120 mL) in a high pressure tube was heated to reflux andstirred for 16 h. The colorless solution turned yellow during reaction.The mixture was then cooled to r.t. and concentrated. The resultingwhite solid was triturated with ethyl ether to give compound 520 (6.5 g,84% yield).

Example 113. Synthesis of Compound 521

A solution of compound 520 (9.93 g, 32.2 mmol) was dissolved in dioxane(15 mL) and treated with concentrated HCl (15 mL) at r.t. for 3 h. Thereaction was concentrated and the resulting solid was collected byfiltration, with washing of the filter cake with EtOAc. The solid wasdried in an oven (50° C.) overnight to give compound 521 (6.94 g, 88%yield).

Example 114. Synthesis of Compound 522

To a solution of compound 521 (0.85 g, 3.47 mmol) in THF (10 mL) wasadded POCl₃ (162 μL, 1.73 mmol) at −10 OC, followed by TEA (966 μL, 6.95mmol). The reaction was stirred at −10° C. for 3 h, and then thesolution was diluted with DCM (20 mL) and filtered over Celite, thefiltrate was concentrated to give compound 522, which was used in thenext step directly. ESI m/z calcd for C₂₀H₂₃ClN₄O₇P [M+H]⁺: 497.09,found 497.09.

Example 115. Synthesis of Compound 523

Compound 522 (0.50 g, 1.0 mmol) and DIPEA (0.4 mL, 2.4 mmol) weredissolved in DCM (5.0 mL) at 0° C., and then compound 401 (0.23 g, 1.0mmol) was added. The reaction was stirred at 0° C. for 2.5 h, thenconcentrated and purified by SiO₂ column to give the title product 523(0.30 g, 43%). ESI m/z calcd for C₃₁H₄₅N₅O₁₁P [M+H]⁺: 694.28, found694.28.

Example 116. Synthesis of Compound 524

Compound 523 (0.30 g, 0.5 mmol) was dissolved in DCM (3 mL), and treatedwith TFA (3 mL) at r.t. for 2 h, then concentrated to give compound 524,which was used in the next step without further purification.

Example 117. Synthesis of Compound 525

Compound 524 (40 mg, 0.063 mmol), compound 110 (40 mg, 0.10 mmol), HATU(24 mg, 0.063 mmol) were dissolved in DCM (5 mL), and then TEA (27.8 μL,0.2 mmol) was added. The reaction mixture was stirred at r.t. overnight.Then the solvent was removed under reduced pressure and the residue waspurified by SiO₂ column to give the title product 525 (53.4 mg, 84%yield). ESI m/z calcd for C₄₈H₆₉N₇O₁₅P [M+H]⁺: 1014.45, found 1014.45.

Example 118. Synthesis of Compound 526

Compound 525 (53.4 mg, 0.053 mmol) was dissolved in DCM (2 mL), andtreated with TFA (2 mL) at r.t. for 2 h, then concentrated to givecompound 526, which was used in the next step without furtherpurification.

Example 119. Synthesis of Compound 527

To a solution of compound 526 (45.0 mg, 0.053 mmol) in DMA (1 mL) wereadded pentafluorophenyl ester 41a (37.0 mg, 0.053 mmol) and DIPEA (17μL, 0.1 mmol). The reaction was stirred overnight and concentrated. Theresidue was purified by prep-HPLC with a gradient of MeCN/H₂O to givethe title product 527 (26.2 mg, 36% yield). ESI m/z calcd forC₆₄H₉₃N₁₁O₁₈PS [M+H]⁺: 1366.61, found 1366.61.

Example 120. Synthesis of Compound 528

Compound 527 (8.0 mg, 0.0058 mmol) was dissolved in toluene (5.0 mL) andheated to reflux overnight, then concentrated and purified by prep-HPLCwith a gradient of MeCN/H₂O to give the title product 528 (6.4 mg, 90%yield). ESI m/z calcd for C₅₆H₈₅N₁₁O₁₆PS [M+H]⁺: 1230.56, found 1230.56.

Example 121. Synthesis of Compound 529

To a solution of tert-butyl3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoate (5.00 g, 12.1 mmol) in10 mL DCM was added 5 mL of TFA. The reaction mixture was stirred atr.t. for 1 h, and then concentrated. The crude product was dissolved inDCM (50 mL), to which NHS (4.25 g, 37 mmol) and EDCI (7.10 g, 37 mmol)were added. The reaction mixture was stirred at r.t. overnight, thenconcentrated and purified by SiO₂ column with a gradient of DCM/MeOH togive the title compound 529 (5.00 g, 91%). ESI m/z calcd for C₂₅H₃₁N₂O₆[M+H]⁺: 455.21, found 455.21.

Example 122. Synthesis of Compound 530

To a solution of compound 110 (1.00 g, 2.5 mmol, 1.0 eq.) in EtOH (10mL) were added compound 529 (1.80 g, 3.9 mmol, 1.5 eq.) and 0.1M NaH₂PO₄(2 mL) at r.t. The reaction mixture was stirred at r.t. overnight, andthen concentrated. The residue was diluted with H₂O (100 mL), thenextracted with EtOAc (3×50 mL). The combined the organic layers weredried over Na₂SO₄, filtered and concentrated, purified by SiO₂ columnwith a gradient of DCM/MeOH to give the title compound 530 (0.93 g,50%). ESI m/z calcd for C₄₂H₆₀N₃O₈ [M+H]⁺: 734.43, found 734.43.

Example 123. Synthesis of Compound 531

In a hydrogenation bottle, Pd/C (0.093 g, 10 wt %) was added to asolution of compound 530 (0.93 g, 1.27 mmol) in EtOAc (20 mL). Themixture was shaken overnight under 1 atm H₂ then filtered through Celite(filter aid), the filtrate was concentrated to afford compound 531 (0.57g, 81%) and used in the next step without further purification. ESI m/zcalcd for C₂₈H₄₈N₃O₈ [M+H]⁺: 554.34, found 554.34.

Example 124. Synthesis of Compound 537

HATU (39.9 g, 105 mmol) was added to a solution of4-(((benzyloxy)carbonyl)amino) butanoic acid (26.1 g, 110 mmol) in DMF(300 mL). After stirring at r.t. for 30 min, the mixture was added to asolution of compound 110 (39.4 g, 100 mmol) and TEA (20.2 g, 200 mmol)in DMF (300 mL). The resulting mixture was stirred at r.t. for 2 h.Water was then added, extracted with EtOAc, the organic layer was washedwith brine, dried over Na₂SO₄. Purification by column chromatography(20% to 70% EA/PE) yielded the title product as a white solid (45 g, 73%yield). ESI m/z calcd for C₃₃H₄₈N₃O₈ [M+H]⁺: 614.34, found 614.15.

Example 125. Synthesis of Compound 538

Compound 537 (100 g, 163 mmol) was dissolved in methanol (500 mL) andhydrogenated (1 atm) with Pd/C catalyst (10 wt %, 10 g) at r.t.overnight. The catalyst was filtered off and the filtrate wereconcentrated under reduced pressure to afford compound 538 (75.8 g, 97%yield) as a brown foamy solid. ¹H NMR (400 MHz, CDCl3) δ 7.11 (s, 1H),6.83 (d, J=10.3 Hz, 2H), 5.04-4.52 (m, 6H), 3.90-3.56 (m, 1H), 2.81 (d,J=5.3 Hz, 2H), 2.63 (dd, J=12.5, 6.1 Hz, 2H), 2.54-2.26 (dd, J=14.0, 7.6Hz, 4H), 1.94-1.64 (m, 3H), 1.44-1.36 (m, 18H), 1.08 (d, J=6.9 Hz, 3H).ESI m/z calcd for C₂₅H₄₂N₃O₆ [M+H]⁺: 480.30, found 480.59.

Example 126. Synthesis of Compound 539

To a solution of compound 537 (1.00 g, 1.63 mmol) in 1 mL DCM was added2 mL TFA, the reaction mixture was stirred at r.t. for 1. h, and thenconcentrated. The resulting crude product 539 was used in the next stepwithout further purification. ESI m/z calcd for C₂₄H₃₂N₃O₆ [M+H]⁺:458.22, found 458.22.

Example 127. Synthesis of Compound 540

To a solution of compound 539 (0.42 g, 0.92 mmol) in DMF (3 mL) wereadded pentafluorophenyl ester 41a (0.63 g, 0.91 mmol) and DIPEA (0.46mL, 2.73 mmol). The reaction was stirred at r.t. overnight, thenconcentrated and purified by SiO₂ column with a gradient of DCM/MeOH togive the title compound 540 (0.67 g, 75% yield) as a yellow oil. ESI m/zcalcd for C₄₉H₇₂N₇O₁₁S [M+H]⁺: 966.49, found 966.49.

Example 128. Synthesis of Compound 541

In a hydrogenation bottle, Pd/C (0.02 g, 10 wt %) was added to asolution of compound 540 (0.40 g, 0.41 mmol) in MeOH (15 mL). a drop of1N HCl was then added to adjust pH to around 4. The mixture was shakenovernight under 1 atm H₂ then filtered through Celite (filter aid), thefiltrate was concentrated to afford compound 541, which was used in thenext step without further purification. ESI m/z calcd for C₄₁H₆₆N₇O₉S[M+H]⁺: 832.46, found 832.46.

Example 129. Synthesis of Compound 587

Compound 110 (0.30 g, 0.76 mmol), compound Z-L-Ala-OH (0.17 g, 0.76mmol) and HATU (0.29 g, 0.76 mmol) were dissolved in DCM (20 mL), towhich TEA (110 μL, 0.8 mmol) was added. The reaction mixture was stirredat r.t. overnight. Then the solvent was removed under reduced pressureand the residue was purified by SiO₂ column to give the title product587 (0.43 g, 95% yield). ESI m/z calcd for C₃₂H₄₆N₃O₈ [M+H]⁺: 600.32,found 600.32.

Example 130. Synthesis of Compound 627

To a solution of H-Lys(Boc)-OH (1.00 g, 3.8 mmol, 1.0 eq.) in EtOH (16mL) was added compound 125 (1.00 g, 5.6 mmol, 1.5 eq.) at r.t. After 0.1M NaH₂PO₄ (3 mL) was added, the reaction mixture was stirred at r.t.overnight. The reaction was concentrated under vacuum, and the residueswas purified by SiO₂ column with a gradient of DCM/MeOH to give thetitle compound 627 (1.62 g, theoretical yield). ESI m/z calcd forC₁₉H₃₀N₃O₇ [M+H]⁺: 412.20, found 412.20.

Example 131. Synthesis of Compound 628

To a solution of carboxylic acid 627 (0.24 g, 0.58 mmol) in EtOAc (10mL) were added pentafluorophenol (0.21 g, 1.17 mmol) and DCC (0.24 g,1.17 mmol). The reaction mixture was stirred at r.t. overnight, and thenfiltered with washing of the filter cake with EtOAc, and the filtratewas concentrated. The resulting PFP-ester (32 mg, 0.056 mmol) wasdissolved in 1 mL DMF, to which compound 531 (50 mg, 0.056 mmol) andi-Pr₂EtN (29 μL, 0.168 mmol) were added. The reaction mixture wasstirred at r.t. for 2 h and concentrated. The residue was purified byHPLC with a gradient of MeCN/H₂O to give the title compound 628 (3 mg,4% yield). ESI m/z calcd for C₆₃H₉₉N₁₀O₁₇S [M+H]⁺: 1299.68, found1299.68.

Example 132. Synthesis of Compound 629

To a solution of compound 628 (3 mg, 0.002 mmol) in 0.5 mL DCM was added1 mL TFA, the reaction mixture was stirred at r.t. for 1 h, thenconcentrated. The crude product was purified by HPLC with a gradient ofMeCN/H₂O to give the title compound 629 (1.43 mg, 52% yield). ESI m/zcalcd for C₅₈H₉₁N₁₀O₁₅S [M+H]⁺: 1199.63, found 1199.62.

Example 133. Synthesis of Compound 632

The pentafluorophenyl ester of compound 627 (0.11 g, 0.19 mmol) wasdissolved in 1 mL DMF, to which compound 541 (0.21 g, 0.25 mmol) andi-Pr₂EtN (86 uL, 0.5 mmol) were added. The reaction mixture was stirredat r.t. for 2 h and concentrated. The residue was purified by prep-HPLCwith a gradient of MeCN/H₂O to give the title product 632 (20 mg, 9%).ESI m/z calcd for C₆₀H₉₃N₁₀O₁₅S [M+H]⁺: 1225.65, found 1225.66.

Example 134. Synthesis of Compound 633

To a solution of compound 632 (20 mg, 0.016 mmol) in 1 mL DCM was added2 mL TFA. The reaction mixture was stirred at r.t. for 1 h, thenconcentrated, and the crude product was purified by prep-HPLC with agradient of MeCN/H₂O to give the title compound 633 (8.9 mg, 18% yield).ESI m/z calcd for C₅₅H₈₅N₁₀O₁₃S [M+H]⁺: 1125.59, found 1125.59.

Example 135. Synthesis of Compound 636

To a solution of H-Dap(Boc)-OH (1.00 g, 4.9 mmol, 1.0 eq.) in EtOH (30mL) was added compound 125 (2.00 g, 7.3 mmol, 1.5 eq.) at r.t. Then 0.1MNaH₂PO₄ (6 mL) was added, and the reaction mixture was stirred at r.t.overnight. The solvents were removed under vacuum, and the residues waspurified by SiO₂ column with a gradient of DCM/MeOH to give the titlecompound 636 (1.41 g, 78%). ESI m/z calcd for C₁₆H₂₄N₃O₇ [M+H]⁺: 370.15,found 370.15.

Example 136. Synthesis of Compound 637

To a solution of compound 636 (1.41 g, 3.8 mmol) in 2 mL DCM was added 5mL TFA. The reaction mixture was stirred at r.t. for 1 h, and thenconcentrated. The crude product 637 was used in the next step withoutfurther purification. ESI m/z calcd for C₁₁H₁₆N₃O₅ [M+H]⁺: 270.10, found270.10.

Example 137. Synthesis of Compound 638

To a solution of above compound 637 in EtOH (20 mL) was added compound125 (1.90 g, 6.9 mmol, 1.5 eq.) at r.t. Then 0.1M NaH₂PO₄ (4 mL) wasadded, and the reaction mixture was stirred at r.t. overnight. After thesolvents were removed under vacuum, then the residues was purified byHPLC with a gradient of H₂O/MeCN to give the title compound 638 (0.45 g,22% yield). ESI m/z calcd for C₁₉H₂₃N₄O₈ [M+H]⁺: 435.14, found 435.14.

Example 138. Synthesis of Compound 639

To a solution of compound 638 (0.15 g, 0.34 mmol), compound 538 (0.17 g,0.34 mmol) and HATU (0.16 g, 0.41 mmol) in DMF (2 mL), TEA (95 μL, 0.68mmol) was added. After stirring at r.t. for 1 h, the reaction wasconcentrated under reduced pressure and the residue was purified byprep-HPLC with a gradient of MeCN/H₂O to give the title compound 639 (34mg, 11% yield). ESI m/z calcd for C₄₄H₆₂N₇O₁₃ [M+H]⁺: 896.43, found896.42.

Example 139. Synthesis of Compound 640

To a solution of compound 639 (34 mg, 0.04 mmol) in 0.5 mL DCM was added1 mL TFA. The reaction mixture was stirred at r.t. for 2 h, and thenconcentrated to afford the title compound 640, which was used in thenext step without further purification. ESI m/z calcd for C₃₅H₄₆N₇O₁₁[M+H]⁺: 740.30, found 740.32.

Example 140. Synthesis of Compound 641

To the solution of compound 640 in DMA (2 mL) was addedpentafluorophenyl ester 41a (28 mg, 0.04 mmol), followed by DIPEA (21μL, 0.12 mmol). The reaction was stirred overnight and then concentratedand purified by prep-HPLC with a gradient of MeCN/H₂O to give the titlecompound 641 (14.4 mg, 29%). ESI m/z calcd for C₆₀H₈₆N₁₁O₁₆S [M+H]⁺:1248.59, found 1248.60.

Example 141. Synthesis of Compound 644

To a solution of compound 132 (0.300 g, 0.329 mmol, 1.0 eq.) andtert-butyl (2-aminoethyl)carbamate hydrochloride (0.063 g, 0.395 mmol,1.2 eq.) in anhydrous DCM (30 mL) at 0° C. was added EDCI (0.189 g,0.988 mmol, 3.0 eq.). After stirring for 10 minutes, the reaction waswarmed to room temperature and stirred overnight. The reaction wasdiluted with DCM and washed with water and brine, dried over anhydrousNa₂SO₄, concentrated and purified by SiO₂ column chromatography(DCM/MeOH) to give compound 644 as a yellow foamy solid (0.132 g, 54%yield). ESI m/z calcd for C₅₂H₈₀N₉O₁₂S [M+H]⁺: 1054.6, found: 1054.6.

Example 142. Synthesis of Compound 645

To a solution of compound 644 (0.132 g, 0.125 mmol, 1.0 eq.) in DCM (4.5mL) at r.t. was added TFA (1.5 mL) and stirred for 1 h. The reaction wasdiluted with anhydrous toluene and concentrated, and this operation wasrepeated for three times to give yellow oil which was purified onprep-HPLC (C₁₈ column, mobile phase A: water, mobile phase B:acetonitrile, from 10% of B to 80% of B in 60 min). The fractions werepooled and lyophilized to give compound 645 (111 mg, 93% yield). ESI m/zcalcd for C₄₇H₇₂N₉₀O₁₀S [M+H]⁺: 954.5, found: 954.5.

Example 143. Synthesis of Compound 648

To a solution of compound 645 (0.050 g, 0.0549 mmol, 1.0 eq.) andtert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (0.024g, 0.0824 mmol, 1.5 eq.) in anhydrous DCM (10 mL) at 0° C. was addedEDCI (0.032 g, 0.1647 mmol, 3.0 eq.). After stirring for 10 minutes, thereaction was warmed to r.t. and stirred overnight. The mixture was thendiluted with DCM and washed with water and brine, dried over anhydrousNa₂SO₄, concentrated and purified by SiO₂ column chromatography(DCM/MeOH) to give the title compound as a yellow foamy solid (0.030 g,46% yield). ESI m/z calcd for C₅₈H₉₂N₉O₁₅S [M+H]⁺: 1186.6, found:1186.6.

Example 144. Synthesis of Compound 649

To a solution of compound 648 (0.030 g, 0.0253 mmol, 1.0 eq.) in DCM(3.0 mL) at r.t was added TFA (1.0 mL). The reaction was stirred for 1 hand then diluted with anhydrous toluene and concentrated, this operationwas repeated for three times to give a yellow oil, which was purified onprep-HPLC (C₁₈ column, mobile phase A: water, mobile phase B:acetonitrile, from 10% of B to 80% of B in 60 min). The fractions werepooled and lyophilized to give compound 649 (11.7 mg, 43% yield). ESIm/z calcd for C₅₃H₈₄N₉O₁₃S [M+H]⁺: 1086.6, found: 1086.6.

Example 145. Synthesis of Compound 652

To a solution of N-(2-aminoethyl)ethane-1,2-diamine (28.7 g, 275 mmol,10.0 eq.) and DMAP (0.034 g, 0.000275 mmol, 0.01 eq.) in anhydrous DCM(350 mL) at 0° C. was added Boc₂O (6.0 g, 0.0275 mmol, 1.0 eq.) inanhydrous DCM (100 mL) over 3 h. The reaction was then warmed to r.t.and stirred overnight, concentrated and purified by SiO₂ columnchromatography (DCM/MeOH) to give the title compound as a yellow oil(4.5 g, 80% yield). ESI m/z calcd for C₉H₂₂N₃O₂ [M+H]⁺: 204.2, found:204.2.

Example 146. Synthesis of Compound 653

To a solution of compound 645 (0.060 g, 0.0658 mmol, 1.0 eq.) andtert-butyl (2-((2-aminoethyl)amino)ethyl)carbamate (0.016 g, 0.0790mmol, 1.2 eq.) in anhydrous DCM (6 mL) at 0° C. was added EDCI (0.038 g,0.1974 mmol, 3.0 eq.). After stirring for 10 minutes, the reaction waswarmed to r.t. and stirred overnight. The mixture was concentrated andpurified on prep-HPLC (C₁₈ column, mobile phase A: water, mobile phaseB: acetonitrile, from 10% of B to 80% of B in 60 min). The fractionswere pooled and lyophilized to give the title compound 653 (48 mg, 66%yield). ESI m/z calcd for C₅₄H₈₅N₁₀O₁₂S [M+H]⁺: 1097.6, found: 1097.6.

Example 147. Synthesis of Compound 654

To a solution of compound 653 (0.048 g, 0.0437 mmol, 1.0 eq.) in DCM(3.0 mL) at r.t. was added TFA (1.0 mL). After stirring for 1 h, thereaction was diluted with anhydrous toluene and concentrated, and thisoperation was repeated for three times to give a yellow oil, which waspurified on prep-HPLC (C₁₈ column, mobile phase A: water, mobile phaseB: acetonitrile, from 10% of B to 80% of B in 60 min). The fractionswere pooled and lyophilized to give the title compound 654 (111 mg, 93%yield). ESI m/z calcd for C₄₉H₇₇N₁₀O₁₀S [M+H]⁺: 997.5, found: 997.5.

Example 148. Synthesis of Compound 658

To a solution of compound 645 (0.400 g, 0.439 mmol, 1.0 eq.) andH-Lys(Boc)-O^(t)Bu·HCl (0.135 g, 0.528 mmol, 1.2 eq.) in anhydrous DCM(40 mL) at 0° C. was added EDCI (0.189 g, 1.317 mmol, 3.0 eq.). Afterstirring for 10 min, the reaction was warmed to r.t. and stirredovernight. The mixture was diluted with DCM and washed with water andbrine, dried over anhydrous Na₂SO₄, concentrated and purified by SiO₂column chromatography (DCM/MeOH) to give compound 658 as a yellow oil(0.43 g, 82% yield). ESI m/z calcd for C₆₀H₉₄N₉O₁₄S [M+H]⁺: 1196.7,found: 1196.7.

Example 149. Synthesis of Compound 559

To a solution of compound 658 (0.230 g, 0.192 mmol, 1.0 eq.) in DCM (6.0mL) at r.t. was added TFA (2.0 mL) and the reaction was stirred for 3 hand then diluted with toluene and concentrated, this operation wasrepeated for three times to give a yellow oil, which was purified onprep-HPLC (C₁₈ column, mobile phase A: water, mobile phase B:acetonitrile, from 10% of B to 80% of B in 60 min). The fractions werepooled and lyophilized to give the title compound (153 mg, 76% yield).ESI m/z calcd for C51H₇₈N₉O₁₂S [M+H]⁺: 1040.5, found: 1040.5.

Example 150. Synthesis of Compound 662

To a solution of compound 658 (0.200 g, 0.167 mmol, 1.0 eq.) andBoc-L-Lys(Boc)-OH (0.070 g, 0.200 mmol, 1.2 eq.) in anhydrous DCM (10mL) at 0° C. was added HATU (0.095 g, 0.250 mmol, 1.5 eq.) and TEA (46μL, 0.334 mmol, 2.0 eq.). The reaction was stirred for 10 min at 0° C.and stirred for 10 minutes, then warmed to r.t. and stirred overnight.The mixture was diluted with DCM and washed with water and brine, driedover anhydrous Na₂SO₄, concentrated and purified by SiO₂ columnchromatography (DCM/MeOH) to give compound 662 as a colourless oil(0.270 g, theoretical yield). ESI m/z calcd for C76H₁₂₂N₁₁O₁₉S [M+H]⁺:1524.9, found: 1524.9.

Example 151. Synthesis of Compound 663

To a solution of compound 662 (0.270 g, 0.177 mmol, 1.0 eq.) in DCM (6.0mL) at r.t. was added TFA (2.0 mL) and stirred for 4 h. The mixture wasdiluted with anhydrous toluene and concentrated, this operation wasrepeated for three times to give yellow oil, which was purified onprep-HPLC (C₁₈ column, mobile phase A: water, mobile phase B:acetonitrile, from 10% of B to 80% of B in 60 min). The fractions werepooled and lyophilized to give the title compound (172 mg, 83% yield).ESI m/z calcd for C₅₇H₉₀N₁₁O₁₃S [M+H]⁺: 1168.6, found: 1168.6.

Example 152. Synthesis of Compound 666

To a solution of ethane-1,2-diamine (30.0 g, 0.5 mol, 10.0 eq.) inanhydrous DCM (500 mL) at 0° C. was added CbzCl (8.53 g, 0.050 mol, 1.0eq.) in anhydrous DCM (250 mL) over 7 h. The reaction was then warmed tor.t. and stirred overnight. The mixture was washed with water and brine,dried over anhydrous Na₂SO₄, and concentrated to give benzyl(2-aminoethyl)carbamate as a white solid (7.0 g, 94% yield). ESI m/zcalcd for C₁₀H₁₄N₂O₂ [M+H]⁺: 195.1, found: 195.2.

Example 153. Synthesis of Compound 667

To a solution of compound 666 (7.0 g, 35.8 mmol, 1.0 eq.) and 37% HCHO(aq) (14 mL, 0.1772 mmol, 5.0 eq.) in MeOH (120 mL) at 0° C. was addedNaBH₃CN (3.9 g, 0.0620 mol, 1.6 eq.), then HOAc (3 mL) was added toadjust pH˜7.0. The mixture was warmed to r.t. and stirred overnight,then concentrated. The residue was dissolved in DCM (200 mL), and washedwith water and brine, dried over anhydrous Na₂SO₄, concentrated andpurified by SiO₂ column chromatography (DCM/MeOH) to give the titlecompound as a light yellow oil (6.4 g, 80% yield). ESI m/: calcd forC₁₂H₁₈N₂O₂ [M+H]⁺: 224.1, found: 224.1.

Example 154. Synthesis of Compound 668

Compound 667 (3.0 g, 13.4 mmol, 1.0 eq.) and Pd/C (0.3 g, 10% Pd/C, 50%wet) were mixed with HCl (3 mL) and MeOH (100 mL) in a hydrogenationbottle and shaken at 100 psi H₂ atmosphere for 5 h. Then the mixture wasfiltered over Celite and the filtrate was concentrated to give the titlecompound as a yellow solid (2.1 g, 98% yield). ¹H NMR (400 MHz, D₂O) δ3.33 (d, J=4.6 Hz, 2H), 3.27 (s, 2H), 2.79 (s, 6H).

Example 155. Synthesis of Compound 669

To a solution of compound 103 (0.58 g, 1.58 mmol, 1.0 eq.) and compound668 (0.051 g, 3.15 mmol, 2.0 eq.) in anhydrous DMF (10 mL) at 0° C. wereadded HATU (0.090 g, 2.37 mmol, 1.5 eq.) and TEA (0.656 mL, 4.74 mmol,3.0 eq.). After stirring for 10 minutes, the reaction was warmed to r.t.and stirred for 90 minutes. The mixture was diluted with H₂O andextracted with EA (3×100 mL). The combined organic layers were washedwith water and brine, dried over anhydrous Na₂SO₄, concentrated to givethe title compound as a yellow foamy solid (0.67 g, 97% yield). ESI m/zcalcd for C₂₁H₃₅N₄O₆ [M+H]⁺: 439.2, found: 439.2.

Example 156. Synthesis of Compound 670

Pd/C (0.2 g, 10% Pd/C, 50% wet) was added to a solution of compound 669(0.60 g, 13.7 mmol, 1.0 eq.) in EA (10 mL). The mixture was shaken at100 psi H₂ atmosphere for 4 h. Then the mixture was filtered over Celiteand the filtrate was concentrated to give the title compound as greenoil (5.50 g, 98% yield). ESI m/z calcd for C₂₁H₃₇N₄O₆₄ [M+H]⁺: 409.3,found: 409.3.

Example 157. Synthesis of Compound 671

To a solution of compound 670 (0.50 g, 1.22 mmol, 1.0 eq.) in 95% EtOH(10 mL) and 0.1M NaH₂PO₄ (2 mL) was added compound 125 (0.683 g, 2.44mmol, 2.0 eq.) and the reaction was stirred overnight and thenconcentrated and purified by SiO₂ column chromatography (DCM/MeOH) togive the title compound as a light yellow oil (0.624 g, 89% yield). ESIm/z calcd for C₂₉H₄₄N₅O₇ [M+H]⁺: 574.3, found: 574.3.

Example 158. Synthesis of Compound 672

To a solution of compound 671 (0.20 g, 0.349 mmol, 1.0 eq) in DCM (6.0mL) at r.t. was added TFA (2.0 mL) and the reaction was stirred for 2 h,then diluted with anhydrous toluene and concentrated, this operation wasrepeated for three times to give the title compound as a yellow oil (165mg, theoretical yield). ESI m/z calcd for C₂₄H₃₆N₅O₅ [M+H]⁺: 474.3,found: 474.3.

Example 159. Synthesis of Compound 673

To a solution of compound 672 (0.165 g, 0.349 mmol, 1.0 eq.) inanhydrous DMF (2 mL) at 0° C. was added compound 41a (0.290 g, 1.047mmol, 1.2 eq.) in anhydrous DMF (3 mL) and the reaction was stirred for10 minutes, then warmed to r.t. and stirred for 1 h. The reactionmixture was concentrated and purified on prep-HPLC (C₁₈ column, mobilephase A: water, mobile phase B: acetonitrile, from 10% of B to 80% of Bin 60 min). The fractions were pooled and lyophilized to give the titlecompound (58 mg, 17% yield) as a light yellow foamy solid. ESI m/z calcdfor C₄₉H₇₆N₉O₁₀S [M+H]⁺: 982.5, found: 982.5.

Example 160. Synthesis of Compound 704

(S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanoic acid (33 mg,0.14 mmol), DCC (32 mg, 0.154 mmol) and pentafluorophenol (39 mg, 0.21mmol) were dissolved in ethyl acetate (20 mL) and the reaction wasstirred at room temperature overnight. The reaction was thenconcentrated to dryness to give compound (S)-perfluorophenyl2-((tert-butoxycarbonyl) (methyl)amino)-3-methylbutanoate, which wasdissolved in 2 mL of DMA, and a solution of compound 2-((1R,3R)-3-((2S,3S)-2-amino-N,3-dimethylpentanamido)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylicacid (52 mg, 0.14 mmol) in 3 mL of DMA and DIPEA (48.5 μL, 0.28 mmol)were added. The reaction was stirred at room temperature overnight andthen concentrated. The residue was diluted with 1 mL of acetonitrile andpurified by reverse phase HPLC with a gradient of MeCN/H₂O to affordcompound 704 (40.2 mg, 49% yield). ESI: m/z: calcd for C₂₈H₄₉N₄O₇S[M+H]⁺: 585.32, found 585.32.

Example 161. Synthesis of Compound 705

To a solution of compound 704 (40 mg, 0.069 mmol) in pyridine (8 mL) at0° C. was added acetic anhydride (20.4 mg, 0.2 mmol), and the reactionwas warmed to room temperature and stirred overnight, then concentrated.The residue was purified by column chromatography (MeOH/DCM) to affordthe title compound 705 (48.1 mg, —100% yield). ESI: m/z: calcd forC₃₀H₅₁N₄O₈S [M+H]⁺: 627.33, found 627.33.

Example 162. Synthesis of Compound 708

Compound 705 (48.1 mg, 0.077 mmol) DCC (17.4 mg, 0.085 mmol) andpentafluorophenol (21.2 mg, 0.115 mmol) were dissolved in ethyl acetate(10 mL) and the reaction was stirred overnight at room temperature, thenconcentrated to dryness to give compound perfluorophenyl2-((6S,9S,12R,14R)-9-((S)-sec-butyl)-6,12-diisopropyl-2,2,5,11-tetramethyl-4,7,10,16-tetraoxo-3,15-dioxa-5,8,11-triazaheptadecan-14-yl)thiazole-4-carboxylate,which was dissolved in 4 mL of DMA, and a solution of compound(4R)-4-amino-2-methyl-5-phenylpentanoic acid, trifluoroacetic acid salt(20.7 mg, 0.1 mmol) in 3 mL of DMA and DIPEA (26.8 μL, 0.154 mmol) wereadded. The reaction was stirred at room temperature overnight and thenconcentrated. The residue was diluted with 1 mL of acetonitrile andpurified by reverse phase HPLC with a gradient of MeCN/H₂O to affordcompound 708 (33 mg, 84% yield). ESI: m/z: calcd for C₄₂H₆₆N₅O₉S [M+H]⁺:816.45, found 816.45.

Example 163. Synthesis of Compound 709

Compound 708 from previous step was dissolved in DCM (1 mL) and treatedwith TFA (1 mL) at r.t. for 2 h. The reaction was concentrated and theresidue was dissolved in EtOH (20 mL). Compound 125 (30.8 mg, 0.11 mmol)and 0.1 M NaH₂PO₄ (4 mL) were added and the resulting mixture wasstirred at r.t. overnight, then concentrated and the residue waspurified by column chromatography (MeOH/DCM) to afford the titlecompound 709 (28.5 mg, 42% yield). ESI m/z: calcd for C₄₅H₆₅N₆O₁₀S[M+H]⁺: 881.44, found 881.44.

Example 164. Synthesis of Compound 712

To a solution of compound 708 (63 mg, 0.077 mmol) in DCM (1 mL) wastreated with TFA (1 mL) at room temperature for 2 h, then concentratedand the residue was dissolved in DMA (4 mL). Compound 711 (65.8 mg, 0.11mmol) and DIPEA (27 μL, 0.154 mmol) were added and the reaction wasstirred at room temperature overnight, then concentrated and the residuewas purified by reverse phase HPLC with a gradient of MeCN/H₂O to affordcompound 712 (14 mg, 16% yield). ESI: m/z: calcd for C₅₅H₈₄N₇O₁₆S[M+H]⁺: 1130.56, found 1130.57.

Example 165. Synthesis of tert-butyl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-14-oxo-4,7,10-trioxa-13-azaheptadecan-1-oate(compound 157a)

Compound 444 (27.92 g, 0.1 mol) and compound 124 (18.3 g, 0.1 mol) wasdissolved in DCM (300 mL), to which DIPEA (12.9 g, 0.1 mol) and EDC(38.6 g, 0.2 mol) were added. The resulting solution was stirred at r.t.overnight and then washed with brine, dried over Na₂SO₄. Filtration,concentration and purification by column chromatography (5% EtOAc/DCM to20% EtOAc/DCM) yielded compound 157a (38.03 g, 86% yield). MS ESI m/zcalcd for C₂₁H₃₅N₂O₈ [M+H]⁺ 443.2394, found 443.2412.

Example 166. Synthesis of17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-14-oxo-4,7,10-trioxa-13-azaheptadecan-1-oicacid (compound 158a)

To a solution of compound 157a (38.0 g, 85.9 mmol) in 150 mL DCM wasadded 50 mL TFA. The reaction mixture was stirred at r.t. for 1 h, andthen diluted with toluene (50 ml), concentrated in vacuo andpurification by column chromatography (10% MeOH/DCM to 20% MeOH/DCM)yielded compound 158a (27.53 g, 83% yield). MS ESI m/z calcd forC₁₇H₂₇N₂O₈ [M+H]⁺ 387.1768, found 387.1792.

Example 167. Synthesis of 2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-14-oxo-4,7,10-trioxa-13-azaheptadecan-1-oate(compound 159a)

NHS (3.20 g, 27.82 mmol) and EDCI (9.70 g, 50.4 mmol) were added to asolution of compound 158a (10.10 g, 26.15 mmol) in DCM (80 mL). Afterstirring at r.t. overnight, the reaction mixture was concentrated andpurified by column chromatography (5 to 20% EtOAc/DCM) to affordcompound 159a (10.73 g, 85% yield). MS ESI m/z calcd for C₂₁H₃₀N₃O₁₀[M+H]⁺ 484.1942, found 484.1978.

Example 168. Synthesis of(2S,5S)-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5-dimethyl-4,7,20-trioxo-10,13,16-trioxa-3,6,19-triazatricosan-1-oicacid (compound 162a)

Compound 161 ((S)-Ala-(S)-Ala, 5.01 g, 31.2 mmol) in the mixture ofethanol (100 ml) and pH 7.5 buffer (0.1 M NaH₂PO₄/Na₂HPO₄, 100 ml), wasadded compound 159a (15.08 g, 31.20 mmol) in four portions in 2 hours.After addition, the mixture was continued to stir for 4 hours,concentrated in vacuo, and purified by column chromatography (10 to 20%MeOH/DCM) to afford compound 162a (13.18 g, 80% yield). MS ESI m/z calcdfor C₂₃H₃₇N₄O₁₀ [M+H]⁺ 529.2511, found 529.2545.

Example 169. Synthesis of (2S,5S)-2,5-dioxopyrrolidin-1-yl23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5-dimethyl-4,7,20-trioxo-10,13,16-trioxa-3,6,19-triazatricosan-1-oate(163a)

NHS (1.60 g, 13.91 mmol) and EDCI (5.90 g, 30.7 mmol) were added to asolution of compound 162a (6.50 g, 12.30 mmol) in DCM (70 mL). Afterstirring at r.t. overnight, the reaction mixture was concentrated andpurified by column chromatography (5 to 20% EtOAc/DCM) to affordcompound 163a (6.61 g, 86% yield). MS ESI m/z calcd for C₂₇H₄₀N₅O₁₂[M+H]⁺ 626.2672, found 626.2698.

Example 170. Synthesis of(2S,4R)-4-((tert-butoxycarbonyl)amino)-5-(3-((2S,5S)-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5-dimethyl-4,7,20-trioxo-10,13,16-trioxa-3,6,19-triazatricosanamido)-4-hydroxyphenyl)-2-methylpentanoicacid (164a)

Compound 121 (3.57 g, 10.55 mmol) in the mixture of ethanol (70 ml) andpH 7.5 buffer (0.1 M NaH₂PO₄/Na₂HPO₄, 60 ml), was added compound 163a(6.60 g, 10.55 mmol) in four portions in 2 hours. After addition, themixture was continued to stir for 4 hours, concentrated in vacuo, andpurified by column chromatography (15 to 25% MeOH/DCM) to affordcompound 164a (7.25 g, 81% yield). MS ESI m/z calcd for C₄₀H₆₁N₆O₁₄[M+H]⁺ 849.4267, found 849.4295.

Example 171. Synthesis of (2R,4S)-4-carboxy-1-(3-((2S,5S)-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5-dimethyl-4,7,20-trioxo-10,13,16-trioxa-3,6,19-triazatricosanamido)-4-hydroxyphenyl)pentan-2-aminium,trifluoroacetate (165a)

To a solution of compound 164a (0.20 g, 0.349 mmol, 1.0 eq) in DCM (6.0mL) at r.t. was added TFA (2.0 mL) and the reaction was stirred for 2 h,then diluted with anhydrous toluene and concentrated, this operation wasrepeated for three times to give the title compound as a yellow oil (165mg, theoretical yield) for the next step without further purification.ESI m/z calcd for C₃₅H₅₄N₆O₁₂ [M+H]⁺: 750.3795, found: 750.3825.

Example 172. Synthesis of(2S,4R)-4-(2-((6S,9R,11R)-6-((S)-sec-butyl)-9-isopropyl-2,3,3,8-tetramethyl-4,7,13-trioxo-12-oxa-2,5,8-triazatetradecan-11-yl)thiazole-4-carboxamido)-5-(3-((2S,5S)-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5-dimethyl-4,7,20-trioxo-10,13,16-trioxa-3,6,19-triazatricosanamido)-4-hydroxyphenyl)-2-methylpentanoicacid (166a)

Compound 165a (45 mg, 0.0600 mmol) and perfluorophenyl ester 41a (45 mg,0.0650 mmol) were dissolved in DMA (5 mL). To the mixture, DIPEA (20 mg,0.154 mmol) was added. The resulting mixture was stirred at r.t.overnight, concentrated and purified by preparative HPLC (C₁₈ column,10-90% MeCN/H₂O) to afford compound 166a (49.1 mg, 65% yield). ESI m/zcalcd for C₆₀H₉₃N₁₀O₁₇S [M+H]⁺: 1256.6442, found: 1256.6510.

Example 173. Synthesis of perfluorophenyl2-methyl-2-(pyrrolidin-1-yl)propanoate 713

2-Methyl-2-(pyrrolidin-1-yl)propanoic acid 25 (401 mg, 2.50 mmol), EDC(654 mg, 3.40 mmol) and pentafluorophenol (480 mg, 2.60 mmol) weredissolved in dichloromethane (45 mL) and the reaction was stirredovernight at room temperature, then concentrated to dryness to givecompound perfluorophenyl 2-methyl-2-(pyrrolidin-1-yl)propanoate 713 (662mg, 82% yield). MS ESI m/z calcd for C₁₄H₁₅F₅NO₂ [M+H]⁺ 324.1024, found324.1045.

Example 174. Synthesis of ethyl2-((5R,7R,10S)-10-((S)-sec-butyl)-3,3-diethyl-7-isopropyl-8,13-dimethyl-9,12-dioxo-13-(pyrrolidin-1-yl)-4-oxa-8,11-diaza-3-silatetradecan-5-yl)thiazole-4-carboxylate714

To the EtOAc solution (40 ml) of pentafluorophenyl ester 713 (650 mg,2.01 mmol), compound 16 (1.085 g, 2.01 mmol) and dry Pd/C (10 wt %, 100mg) were added. The reaction mixture was stirred under hydrogenatmosphere (1 atm) for 24 h, and then filtered through a plug of Celite,with washing of the filter pad with EtOAc. The combined organic portionswere concentrated and purified by column chromatography with a gradientof 0-5% methanol in CH₂Cl₂ to deliver compound 714 (1.10 g, 84% yield).MS ESI m/z calcd for C₃₃H₆₁N₄O₅SSi [M+H]⁺ 653.4133, found 653.4148.

Example 175. Synthesis of ethyl2-((1R,3R)-3-((2S,3S)—N,3-dimethyl-2-(2-methyl-2-(pyrrolidin-1-yl)propanamido)pentanamido)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate,715

Compound 714 (1.10 g, 1.68 mmol) was dissolved in AcOH/water/THF (v/v/v3:1:1, 25 mL), and stirred at r.t. for 2 days. After the reaction wasconcentrated, toluene was added and concentrated again; this step wasrepeated two times to afford compound 715, which was used directly inthe next step. MS ESI m/z calcd for C₂₇H₄₇N₄O₅S [M+H]⁺ 539.3268, found539.3295.

Example 176. Synthesis of 2-((1R,3R)-3-((2S,3S)—N,3-dimethyl-2-(2-methyl-2-(pyrrolidin-1-yl)propanamido)pentanamido)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylicacid, 716

An aqueous solution of LiOH (0.4 N, 5 mL) was added to a solution ofcompound 715 (0.85 g, 1.65 mmol) in MeOH (20 mL) at 0° C. The reactionmixture was stirred at r.t. for 2 h and then concentrated. Columnchromatography (pure CH₂Cl₂ to 80:20:1 CH₂Cl₂/MeOH/NH₄OH) affordedcompound 716 (773 mg, 90% yield for two steps) as an amorphous solid. MSESI m/z calcd for C₂₅H₄₃N₄O₅S [M+H]⁺ 511.2955, found 511.2980.

Example 177. Synthesis of 2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-(2-methyl-2-(pyrrolidin-1-yl)propanamido)pentanamido)-4-methylpentyl)thiazole-4-carboxylicacid, 717

A solution of compound 716 (765 mg, 1.50 mmol) and DMAP (180 mg, 1.48mmol) in anhydrous THF (30 mL) and anhydrous DMF (15 mL) was cooled to0° C., to which DIPEA (3.0 mL, 17.2 mmol) and acetic anhydride (1.0 g,9.79 mmol) were added. The reaction mixture was allowed to warm to r.t.and stirred for 4 h, and then concentrated. Column chromatography (5-50%MeOH/DCM) delivered compound 717 (785 mg, 95% yield) as an amorphoussolid. MS ESI m/z calcd for C₂₇H₄₅N₄O₆S [M+H]⁺ 553.3061, found 553.3095.

Example 178. Synthesis of perfluorophenyl 2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-(2-methyl-2-(pyrrolidin-1-yl)propanamido)pentanamido)-4-methylpentyl)thiazole-4-carboxylate,718

To a solution of compound 717 (775 mg, 1.40 mmol) in anhydrous DCM (10mL) was added EDC (805 mg, 4.19 mmol) and pentafluorophenol (276 mg,1.50 mmol) at room temperature under N₂. The mixture was stirred at roomtemperature for 4 h, and then diluted in DCM (100 mL), washed with water(2×200 mL) and brine (200 mL), dried over anhydrous sodium sulfate,filtered, concentrated and purified by SiO₂ column chromatography (50%EtOAc/PE) to give compound 718 as a white solid (815 mg, 81% yield) MSESI m/z calcd for C₃₃H₄₄F₅N₄O₆S [M+H]⁺: 719.2901, found: 719.2945.

Example 179. Synthesis of (2S,4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-(2-methyl-2-(pyrrolidin-1-yl)propanamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-((2S,5S)-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5-dimethyl-4,7,20-trioxo-10,13,16-trioxa-3,6,19-triazatricosanamido)-4-hydroxyphenyl)-2-methylpentanoicacid

In a solution of compound 718 (153 mg, 0.213 mmol) and compound 165a(160 mg, 0.213 mmol) in 7 mL of DMA, DIPEA (100 μL, 0.575 mmol) wereadded. The reaction was stirred at room temperature overnight and thenconcentrated. The residue was diluted with 1 mL of acetonitrile andpurified by reverse phase HPLC with a gradient of MeCN/H₂O (10% MeCN to40% MeCN in 45 min, d2 cm×L25 cm, C-18 column, 8 ml/min) to affordcompound 719 (166.1 mg, 61% yield). ESI: m/z: calcd for C₆₂H₉₅N₁₀O₁₇S[M+H]⁺: 1282.6598, found 1282.6630.

Example 180. Synthesis of(2S,4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3-((2S,5S)-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5-dimethyl-4,7,20-trioxo-10,13,16-trioxa-3,6,19-triazatricosanamido)-4-hydroxyphenyl)-2-methylpentanoicacid, 720

Compound 33 (30.2 mg, 0.056 mmol), EDC (25.0 mg, 0.130 mmol) andpentafluorophenol (11 mg, 0.060 mmol) were dissolved in dichloromethane(4 mL) and the reaction was stirred overnight at room temperature, thenconcentrated to dryness to give compound perfluorophenyl2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxylate,33a, which was dissolved in 4 mL of DMA, and a solution of compound 165a(160 mg, 0.213 mmol) in 3 mL of DMA and DIPEA (26.8 μL, 0.154 mmol) wereadded. The reaction was stirred at room temperature overnight and thenconcentrated. The residue was diluted with 1 mL of acetonitrile andpurified by reverse phase HPLC with a gradient of MeCN/H₂O (10% MeCN to40% MeCN in 45 min, d2 cm×L25 cm, C-18 column, 8 ml/min) to affordcompound 720 (133.1 mg, 48% yield). ESI: m/z: calcd for C₆₁H₉₃N₁₀O₁₇S[M+H]⁺: 1269.6442, found 1282.6630.

Example 181. Synthesis of (2S,4R)-tert-butyl4-((tert-butoxycarbonyl)amino)-5-(4-((2S,5S)-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5-dimethyl-4,7,20-trioxo-10,13,16-trioxa-3,6,19-triazatricosanamido)phenyl)-2-methylpentanoate,721

Compound 488 (310 mg, 0.82 mmol) in the mixture of ethanol (70 ml) andpH 7.5 buffer (0.1 M NaH₂PO₄/Na₂HPO₄, 60 ml), was added compound 163a(660 mg, 1.055 mmol) in four portions in 2 hours. After addition, themixture was continued to stir for 4 hours, concentrated in vacuo, andpurified by column chromatography (15 to 25% EtOAc/DCM) to affordcompound 714 (604.5 mg, 83% yield). MS ESI m/z calcd for C₄₄H₆₉N₆O₁₃[M+H]+ 889.4923, found 889.4965.

Example 182. Synthesis of (2S,4R)-4-amino-5-(4-((2S,5S)-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5-dimethyl-4,7,20-trioxo-10,13,16-trioxa-3,6,19-triazatricosanamido)phenyl)-2-methylpentanoicacid, trifluoroacetic acid Salt

To a solution of compound 721 (0.20 g, 0.225 mmol, 1.0 eq) in DCM (6.0mL) at r.t. was added TFA (2.0 mL) and the reaction was stirred for 2 h,then diluted with anhydrous toluene and concentrated, this operation wasrepeated for three times to give the title compound as a yellow oil (165mg, theoretical yield) for the next step without further purification.ESI m/z calcd for C₃₅H₅₃N₆O₁₁ [M+H]⁺: 732.3773, found: 732.3795.

Example 183. Synthesis of (2S,4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(4-((2S,5S)-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,5-dimethyl-4,7,20-trioxo-10,13,16-trioxa-3,6,19-triazatricosanamido)phenyl)-2-methylpentanoicacid (723, as a reference control)

Compound 33 (30.2 mg, 0.056 mmol), EDC (25.0 mg, 0.130 mmol) andpentafluorophenol (11 mg, 0.060 mmol) were dissolved in dichloromethane(4 mL) and the reaction was stirred overnight at room temperature, thenconcentrated to dryness to give compound perfluorophenyl2-((1R,3R)-1-acetoxy-3-((2S,3S)—N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxylate,which was dissolved in 4 mL of DMA, and a solution of compound 722 (160mg, 0.213 mmol) in 3 mL of DMA and DIPEA (26.8 μL, 0.154 mmol) wereadded. The reaction was stirred at room temperature overnight and thenconcentrated. The residue was diluted with 1 mL of acetonitrile andpurified by reverse phase HPLC with a gradient of MeCN/H₂O (10% MeCN to40% MeCN in 45 min, d2 cm×L25 cm, C-18 column, 8 ml/min) to affordcompound 723 (47.7 mg, 68% yield). ESI: m/z: calcd for C₆₁H₉₃N₁₀O₁₆S[M+H]⁺: 1253.6492, found 1253.6540.

Example 184. General method of preparation of antibody conjugates ofcompounds 128, 132, 437, 481, 495, 528, 629, 633, 641, 645, 649, 654,659, 663, 673, 709, 712, 166a, 719, 720 and 723

To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0˜8.0, were addedof 0.70˜2.0 mL PBS buffer of 100 mM NaH₂PO₄, pH 6.5˜8.5 buffers, TCEP(14-35 μL, 20 mM in water) and the compounds 128, 132, 437, 481, 495,528, 629, 633, 641, 645, 649, 654, 659, 663, 673, 709, 712, 166a, 719,720 and 723 (14-28 μL, 20 mM in DMA independently, followed by additionof 4-(azidomethyl)benzoic acid (14-50 μL, 20 mM in pH 7.5, PBS buffer).The mixture was incubated at RT for 4˜18 h, then DHAA (135 μL, 50 mM)was added in. After continuous incubation at RT overnight, the mixturewas purified on G-25 column eluted with 100 mM NaH₂PO₄, 50 mM NaCl pH6.0˜7.5 buffer to afford 12.2˜18.6 mg of the conjugate compounds C-128,C-132, C-437, C-481, C-495, C-528, C-629, C-633, C-641, C-644, C-645,C-649, C-654, C-659, C-663, C-673, C-709, C-712, C-166a, C-719, C-720and C-723, (80%-93% yield) accordingly in 13.4˜15.8 ml of the NaH₂PO₄buffer. The drug/antibody ratio (DAR) was 3.4˜3.9 for the conjugates,wherein DAR was determined via UPLC-QTOF mass spectrum. It was 94-99%monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mmID×30 cm, 0.5 ml/min, 100 min) and a single band measured by SDS-PAGEgel.

Example 185. In vitro cytotoxicity evaluation of conjugate C-128, C-132,C-437, C-481, C-495, C-528, C-629, C-633, C-641, C-644, C-645, C-649,C-654, C-659, C-663, C-673, C-709, C-712, C-166a, C-719, C-720 andC-723, in comparison with T-DM1

The cell line used in the cytotoxicity assays was NCI-N87, a humangastric carcinoma cell line; the cells were grown in RPMI-1640 with 10%FBS. To run the assay, the cells (180 μl, 6000 cells) were added to eachwell in a 96-well plate and incubated for 24 hours at 37° C. with 5%CO₂. Next, the cells were treated with test compounds (20 μl) at variousconcentrations in appropriate cell culture medium (total volume, 0.2mL). The control wells contain cells and the medium but lack the testcompounds. The plates were incubated for 120 hours at 37° C. with 5%CO₂. MTT (5 mg/ml) was then added to the wells (20 μl) and the plateswere incubated for 1.5 hr at 37° C. The medium was carefully removed andDMSO (180 μl) was added afterward. After it was shaken for 15 min, theabsorbance was measured at 490 nm and 570 nm with a reference filter of620 nm. The inhibition % was calculated according to the followingequation: inhibition %=[1-(assay-blank)/(control-blank)]×100. Theresults are listed in Table 1.

TABLE 1. The Structures of the Her2-tubulysin analog conjugates of thepatent application along with their results of the IC₅₀ cytotoxicityagainst NCI-N87 cells: Com- pound # Structures and its IC50 againstNCI-N87 cells 129 (C-128)

133 (C-132)

C-437

C-481

C-495

C-528

C-629

C-633

C-641

C-645

C-649

C-654

C-659

C-663

C-673

C-709

C-712

C-166a

C-719

C-720

C-723

Example 186. Antitumor Activity In Vivo (BALB/c Nude Mice BearingNCI-N87 Xenograft Tumor)

The in vivo efficacy of conjugates C-166a, C-719, C-720, C-723 alongwith T-DM1 were evaluated in a human gastric carcinoma N-87 cell linetumor xenograft models. Five-week-old female BALB/c Nude mice (40animals) were inoculated subcutaneously in the area under the rightshoulder with N-87 carcinoma cells (5×10⁶ cells/mouse) in 0.1 mL ofserum-free medium. The tumors were grown for 7 days to an average sizeof 125 mm³. The animals were then randomly divided into 6 groups (6animals per group). The first group of mice served as the control groupand was treated with the phosphate-buffered saline (PBS) vehicle. Fivegroups were treated with conjugates C-166a, C-719, C-720, C-723 andT-DM1 respectively at dose of 6 mg/Kg administered intravenously. Threedimensions of the tumor were measured every 3 or 4 days (twice a week)and the tumor volumes were calculated using the formula tumor volume=½(length×width×height). The weight of the animals was also measured atthe same time. A mouse was sacrificed when any one of the followingcriteria was met: (1) loss of body weight of more than 20% frompretreatment weight, (2) tumor volume larger than 1500 mm³, (3) too sickto reach food and water, or (4) skin necrosis. A mouse was considered tobe tumor-free if no tumor was palpable. The results were plotted in FIG.22 .

Example 187

The toxicity study of the conjugate in comparison with conjugatesC-166a, C-719, C-720, C-723 and T-DM1. 66 female ICR mice, 6-7 weeksold, were separated into 11 groups. Each group included 6 mice for theliver toxicity study. The first group of mice served as the controlgroup and was treated with the phosphate-buffered saline (PBS) vehicle.10 groups were treated with conjugates C-166a, C-719, C-720, C-723 andT-DM1 respectively at dose of 75 mg/Kg and 150 mg/Kg administeredintravenously. The body weight changes for each animal were measuredevery day for 12 days. The blood collection was followed the NCI'sGuidelines for Rodent Blood Collection. Basically, Blood samples werecollected through retro-orbital sinuses of the mice, and centrifuged toobtain the sera on Day 5 after administration. The levels of aspartateaminotransferase (AST), alanine aminotransferase (ALT) and alkalinephosphatase (ALP) were analyzed using PUS-2018 semi-automaticbiochemistry analyzer with a commercial kid (using aspartate and alanineas substrates, respectively). Reference values were established byfollowing reactive dynamics, according to manufacturer'srecommendations. After blood collection, the mice were sacrificed andthe mice livers were sliced for pathogen studies. The results of AST andALT on average were shown in Table 2, and the results of the animal bodyweight changes on average and the pathogen pictures were shown in FIGS.23A and 23B and FIGS. 24A-24F, respectively.

The results of liver toxicity plus the body weight changes indicatedthat at the much higher dose of 75 mg/Kg and 150 mg/Kg, the conjugatesC-166a, and C-719 were less toxic than T-DM1, the conjugate C-720 hadsomehow the similar toxicity to T-DM1, and the conjugate C-723 was muchmore toxic than T-DM1. The toxicity order at the tested doses was:C-723>T-DM1≥C-720>C-719>C-166a >PBS. Since conjugates C-166a, C-719,C-720, and C-723 had a similar in vivo activities and all of them hadbetter in vivo activity than T-DM1 as indicated in FIGS. 23A and 23B,therefore the therapeutical windows for conjugates C-166a and C-719would be much better than T-DM1. In summary, the replacement ofN-alkyl-piperidine-2-carboxylic group on the left side of tubulysin by2-N-alkyl-2,2-dialkyl-acetic group can dramatically reduce animal sidetoxicity while maintaining the in vivo activities of the tubulysinanalogs.

TABLE 2 The results of AST and ALT on average of the tested animals. AST(IU/L) ALT (IU/L) Day 5 Day 12 Day 5 Day 12 PBS  91.3 ± 11.4  95.9 ±11.0  36.3 ± 18.5  27.9 ± 8.0 T-DM1, 1349.7 ± 321.5  303.2 ± 157.8 154.4± 96.5 164.6 ± 61.4 75 mg/Kg T-DM1, 3276.6 ± 724.4 1509.6 ± 399.3 305.9± 142.9 407.3 ± 53.8 150 mg/Kg C-166a,  173.6 ± 13.1  100.5 ± 16.1  56.8± 13.3  48.3 ± 9.5 75 mg/Kg C-166a,  480.3 ± 50.5  131.5 ± 29.3 126.5 ±38.1  71.7 ± 15.3 150 mg/Kg C-719,  185.6 ± 14.8  111.5 ± 19.2  62.8 ±14.8  52.7 ± 11.9 75 mg/Kg C-719,  543.5 ± 67.5  159.5 ± 38.5 137.5 ±43.7  83.4 ± 19.2 150 mg/Kg C-720,  904.5 ± 231.8  264.4 ± 49.6 145.6 ±60.7 139.8 ± 28.9 75 mg/Kg C-720, 3083.1 ± 803.0 1576.6 ± 34.9 401.8 ±59.0 335.9 ± 41.5 150 mg/Kg C-723, 1673.4 ± 335.5 1093.1 ± 351.6 206.8 ±84.1 196.0 ± 41.6 75 mg/Kg C-723, 4083.4 ± 353.9 1861.8 ± 787.1 587.6 ±111.2 483.9 ± 220.9 150 mg/Kg

What is claimed is:
 1. A conjugate of a cell binding molecule with acytotoxic agent having a structure of Formula (I):

or a pharmaceutically acceptable salt, hydrate, or hydrated saltthereof; or a polymorphic crystalline structure thereof, or an opticalisomer, racemate, diastereomer or enantiomer thereof; wherein T is atargeting or cell-binding molecule; L is a releasable linker;

is a linkage bond that L connects to an atom inside the bracketindependently; n is 1˜20 and m is 1˜10; wherein T is an antibody; asingle chain antibody; an antibody fragment that binds to the targetcell; a monoclonal antibody; a single chain monoclonal antibody; or amonoclonal antibody fragment that binds the target cell; a chimericantibody; a chimeric antibody fragment that binds to the target cell; adomain antibody; a domain antibody fragment that binds to the targetcell; lymphokine; a hormone; a vitamin; a growth factor; a colonystimulating factor; or a transferrin; a binding peptide, or protein, orantibody, or molecule attached on an albumin, a polymer, a dendrimer, aliposome, a nanoparticle, a vesicle, or a capsid; wherein the linker Lhas the formula: —Ww-(Aa)r-Vv-; wherein: —W— is a Stretcher unit; w is 0or 1; each -Aa-is independently an Amino Acid unit; r is independentlyan integer ranging from 0 to 12; —V— is a Spacer unit; and v is 0, 1 or2; the Stretcher unit (—W—), when present, links a targeted bindingmolecular unit (T) to an amino acid unit (-Aa-), or links V when an Aais not present; the Stretcher unit W independently contains aself-immolative spacer, peptide unit, a hydrazone bond, disulfide orthiolether bond; W linked to T has a structure of:

wherein R²⁰ and R²¹ are selected from —C₁˜C₉ alkylene-, —C₁˜C₇carbocyclo-, —O—(C₁˜C₈ alkyl)-, -arylene-, —C₁˜C₉ alkylene-arylene-,-arylene, —C₁˜C₉ alkylene-, —C₁˜C₉ alkylene-(C₁˜C₈ carbocyclo)-, —(C₃˜C₇carbocyclo)-C₁˜C₉ alkylene-, —C₃˜C₈ heterocyclo-, —C₁˜C₁₀alkylene-(C₃˜C₈ heterocyclo)-, —(C₃-C₈ heterocyclo)-C₁˜C₉ alkylene-,—(CH₂CH₂O)_(k)—, —(CH(CH₃)CH₂O)_(k)—, and —(CH₂CH₂O)_(k)—CH₂—; k is aninteger ranging from 1˜20; R′ and R″ are independently H or CH₃; theSpacer unit (—V—), when present, links an Amino Acid unit to theantimitotic agent when an Amino Acid unit is present or the Spacer unitlinks the Stretcher unit to antimitotic agent when the Amino Acid unitis absent, or the Spacer unit links antimitotic agent to the bindingmolecule (T) when both the Amino Acid unit and Stretcher unit areabsent; the spacer unit contains a function group that substantiallyincreases the water solubility, biological transport, preferential renalclearance, uptake, absorption, biodistribution, and/or bioavailabilityof the conjugate; the Spacer unit includes a self-immolative ornon-self-immolative; the non-self-immolative Spacer unit is one in whichpart or all of the Spacer unit remains bound to the antimitotic agentafter cleavage of the Amino Acid unit from the conjugate; theself-immolative unit includes an aromatic compound that iselectronically similar to a para-aminobenzyl-carbamoyl (PAB) group,2-aminoimidazol-5-methanol group, heterocyclic PAB group,beta-glucuronide, or ortho or para-aminobenzylacetal; or one of thefollowing structures:

wherein the (*) atom is the point of attachment of additional spacer orreleasable linker unit, the antimitotic agent, and/or the bindingmolecule (T); X, Y and Z³ are independently NH, O, or S; Z² is H, NH, Oor S independently; v is 0 or 1; Q is independently H, OH, C₁˜C₆ alkyl,(OCH₂CH₂)_(n) F, Cl, Br, I, OR¹⁷, or SR¹⁷, NR¹⁷R¹⁸, N═NR¹⁷, N═R¹⁷,NR¹⁷R¹⁸, NO₂, SOR¹⁷R¹⁸, SO₂R¹⁷, SO₃R¹⁷, OSO₃R¹⁷, PR¹⁷R¹⁸, POR¹⁷R¹⁸,PO₂R¹⁷R¹⁸, OPO(OR¹⁷)(OR¹⁸), or OCH₂PO(OR¹⁷(OR¹⁸) wherein R¹⁷ and R¹⁸ areindependently H, C₁˜C₈ alkyl; C₂-C₈ alkenyl, alkynyl, or heteroalkyl;C₃˜C aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl,heteroaralkyl, or alkylcarbonyl; or a pharmaceutical cation saltthereof; the non-self-immolative spacer unit having a structure of:

 or L- or D-, natural or unnatural peptides containing 1˜20 the same ordifferent amino acids; wherein the “*” and “

” atom are the point of attachment of additional spacer or releasablelinker, the antimitotic agent, and/or the binding molecule; m is 1˜10; nis 1˜20; X₂, X₃, X₄, X₅, or X₆ are independently NH; NHNH; N(R₁₂);N(R₁₂)N(R_(12′)); O; S; C₁˜C₆ alkyl; C₂˜C₆ of heteroalkyl,alkylcycloalkyl, or heterocycloalkyl; C₃˜C₈ aryl, Ar-alkyl,heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,alkylcarbonyl, or heteroaryl; CH₂OR₁₂, CH₂SR₁₂, CH₂NHR₁₂, or 1˜8 aminoacids; wherein R₁₂ and R_(12′) are independently H; C₁˜C₈ alkyl; C₂˜C₈hetero-alkyl, alkylcycloalkyl, or heterocycloalkyl; C₃˜C₈ aryl,Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,alkylcarbonyl, or heteroaryl; or C₁˜C₈ ester, ether, or amide; orpolyethyleneoxy unit of formula (OCH₂CH₂)_(p) or (OCH₂CH(CH₃))_(p),wherein p is an integer from 0 to about 1000, or a combination of two ormore thereof; a releasable component of the linker L that at least onebond in L can be broken under physiological conditions: a pH-labile,acid-labile, base-labile, oxidatively labile, metabolically labile,biochemically labile or enzyme-labile bond, which having one of thefollowing structures: —(CR₁₅R₁₆)_(m)(Aa)r(CR₁₇R₁₈)_(n)(OCH₂C₁₂)_(t)—,—(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(Aa)_(r)(OCH₂CH₂)_(t)—, -(Aa)_(r)-(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(t)—,—(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(r)(Aa)_(t)-,—(CR₁₅R₁₆)_(m)(CR₁₇═CR₁₈)(CR₁₉R₂₀)_(n)(Aa)_(t)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(NR₂₁CO)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(Aa)_(t)(NR₂₁CO)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(OCO)(Aa)_(t)-(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(OCNR₁₇)(Aa)_(t)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)—(CO)(Aa)_(t)-(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(NR₂₁CO)(Aa)_(t)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)—(OCO)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(OCNR₁₇)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)(CO)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—(CR₁₅R₁₆)_(m)-phenyl-CO(Aa)_(t)-(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(m)-furyl-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—(CR₁₅R₆)_(m)-oxazolyl-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(m)-thiazolyl-CO(Aa)_(t)(CCR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(t)-thienyl-CO(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(t)-imidazolyl-CO—(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(t)-morpholino-CO(Aa)_(t)-(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(t)-piperazino-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(t)—N-methylpiperazin-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—(CR₁₅R₁₆)_(m)-(Aa)_(t)phenyl-, —(CR₁₅R₁₆)_(m)-(Aa)_(t)furyl-,—(CR₁₅R₁₆)_(m)-oxazolyl(Aa)_(t)-, —(CR₁₅R₁₆)_(m)-thiazolyl(Aa)_(t)-,—(CR₁₅R₁₆)_(m)-thienyl-(Aa)_(t)-, —(CR₁₅R₁₆)_(m)-imidazolyl(Aa)_(t)-,—(CR₁₅R₁₆)_(m)-morpholino-(Aa)_(t)-,—(CR₁₅R₁₆)_(m)-piperazino-(Aa)_(t)-,—(CR₁₅R₁₆)_(m)—N-methylpiperazino-(Aa)_(t)-,—K(CR₁₅R₁₆)_(m)(Aa)_(r)(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(t)—,—K(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(Aa)_(r)(OCH₂CH₂)_(t)—,—K(Aa)_(r)-(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(t)—,—K(CR₁₅R₁₆)_(m)(CR₁₇R₁₈)_(n)(OCH₂CH₂)_(r)(Aa)_(t)-,—K(CR₁₅R₁₆)_(m)—(CR₁₇═CR₁₈)(CR₁₉R₂₀)_(n)(Aa)_(t)(OCH₂CH₂)_(r),—K(CR₁₅R₁₆)_(m)(NR₁₁CO)(Aa)_(t)-(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₅R₆)_(m)(Aa)_(t)(NR₂₁CO)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)—(OCO)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)(OCNR₁₇)(Aa)_(t)(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)(CO)(Aa)_(t)- (CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)(NR₂₁CO)(Aa)_(t)-(CR₁₉R₂₀)_(n)—(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)—(OCO)(Aa)_(t)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)—(OCNR₁₇)(Aa)_(t)-(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K—(CR₁₅R₁₆)_(m)(CO)(Aa)_(t)(CR₁₉R₂₀)_(n)(OCH₂CH₂)_(r)—,—K(CR₁₅R₁₆)_(m)-phenyl-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—K—(CR₁₅R₁₆)_(m)-furyl-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(m)-oxazolyl-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(m)-thiazolyl-CO(Aa)_(t)-(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(t)-thienyl-CO(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(t)imidazolyl-CO—(CR₁₇R₁₈)_(n)—,—K(CR₅R₆)_(t)morpholino-CO(Aa)_(t)-(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(t)-piperazino-CO(Aa)_(t)-(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(t)—N-methylpiperazin-CO(Aa)_(t)(CR₁₇R₁₈)_(n)—,—K(CR₁₅R₁₆)_(m)-(Aa)_(t)phenyl, —K—(CR₁₅R₁₆)_(m)-(Aa)_(t)furyl-,—K(CR₁₅R₁₆)_(m)-oxazolyl-(Aa)_(t)-, —K(CR₁₅R₁₆)_(m)-thiazolyl(Aa)_(t)-,—K(CR₁₅R₁₆)_(m)-thienyl-(Aa)_(t)-, —K(CR₁₅R₁₆)_(m)-imidazolyl(Aa)_(t)-,—K(CR₁₅R₁₆)_(m)-morpholino(Aa)_(t)-, —K(CR₁₅R₁₆)_(m)piperazino(Aa)_(t)G,—K(CR₅R₆)_(m)—N-methyl-piperazino(Aa)_(t)-; wherein m, Aa, R₁₃, R₁₄, andR₁₅ are described above; t and r here are 0-100 independently; R₁₆, R₁₇,R₁₈, R₁₉, and R₂₀ are independently H; halide; C₁˜C₈ alkyl orheteroalkyl, C₂˜C₈ aryl, alkenyl, alkynyl, ether, ester, amine or amide,C₃˜C₈ aryl, which optionally substituted by one or more halide, CN,NR₁₂R_(12′), CF₃, OR₁₂, Aryl, heterocycle, S(O)R₁₂, SO₂R₁₂, —CO₂H,—SO₃H, —OR₁₂, —CO₂R₁₂, —CONR₁₂, —PO₂R₁₂R₁₃, —PO₃H or P(O)R₁₂R_(12′)R₁₃;K is NR₁₂, —SS—, —C(═O)—, —C(═O)NH—, —C(═O)O—, —C═NH—O—, —C═N—NH—,—C(═O)NH—NH—, O, S, Se, B, Het (heterocyclic or heteroaromatic ringhaving C₃˜C₁₂); or a peptide containing the same or different 1-20 aminoacids; inside the bracket of the Formula (I) is an antimitotic agentwherein R¹, R², R³, and R⁴ are independently linear or branched C₁-C₈alkyl, or alkylalcohol; C₂-C₈ heteroalkyl, alkylcycloalkyl,heterocycloalkyl, alkyl ether, alkyl carboxylate, alkyl amine, alkylester, or alkyl amide; C₃-C₈ aryl, Ar-alkyl, heterocyclic, carbocyclic,cycloalkyl, heteroalkylcycloalkyl, or alkylcarbonyl; or two R's: R¹R²,R³R⁴, R⁵R⁶, or R¹²R¹³ together form a 3˜7 membered carbocyclic,cycloalkyl, heterocyclic, heterocycloalkyl, aromatic or heteroaromaticring system; Y is N or C; R¹, R², R³, and R⁴ may optionally beindependently absent; wherein R⁵, R⁶, R⁸ and R¹⁰ are independently H,linear or branched C₁-C₄ alkyl or C₂-C₄ heteroalkyl; wherein R⁷ is H,R¹⁴, —R¹⁴C(═O)X¹R¹⁵; or —R¹⁴X¹R¹⁵; X¹ is O, S, S—S, NH, or NR¹⁴; whereinR⁹ is H, —O—, —OR¹⁴, —OC(═O)R¹⁴—, —OC(═O)NHR¹⁴—, —OC(═O)NR¹⁴R¹⁵—,—OC(═O)R¹⁴SSR¹⁵—, OP(═O)(OR¹⁴)—, or OR¹⁴OP(═O)(OR¹⁵); wherein R¹¹ is H,R¹⁴, —R¹⁴C(═O)R¹⁶, —R¹⁴C(═O)X²R¹⁶, —R¹⁴X²R¹⁶, or —R¹⁴C(═O)X², wherein X²is —O—, —S—, —NH—, —NHS(O₂), —NHS(O), —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—,—S(═O)—R¹⁴—, or —NHR¹⁴—, provided that when R¹¹ is —R¹⁴C(═O)X²— andconnects to L, wherein X² is —O—, R¹⁴ is not C₁-C₈ alkyl; wherein R¹² isH, R¹⁴, —O—, —S—, —N—, ═N—, ═NNH—, —OH, —SH, —NH₂, ═NH, ═NNH₂, —NH(R¹⁴),—OR¹⁴, —C(O)O—, —C(O)OR¹⁶—, —COR¹⁶, —COOR¹⁴—, C(O)NH—, C(O)NH₂,C(O)NHR¹⁴, —SR¹⁴, —S(═O)R¹⁴, —P(═O)(OR¹⁶)₂, —OP(═O)(OR¹⁶)₂,—CH₂OP(═O)(OR¹⁶)₂, or —SO₂R¹⁶; wherein R¹³ is linear or branched C₁˜C₁₀alkyl, alkyl acid, alkyl amide, or alkyl amine; or C₂-C₁₀ heteroalkyl;or C₃-C₁₀ Ar; Ar is an aromatic or hetero aromatic group, composed ofone or several rings, comprising four to ten carbon atoms, the heteroaromatic group is an aromatic group that has one or several carbon atomsreplaced by hetero atoms, the aryl or Ar is an aromatic group, whereinone or several H atoms can be replaced independently by R¹⁷, F, Cl, Br,I, OR¹⁶, SR¹⁶, NR¹⁶R¹⁷, N═NR¹⁶, N═R¹⁶, NR¹⁶R¹⁷, NO₂, SOR¹⁶R¹⁷, SO₂R¹⁶,SO₃R¹⁶, OSO₃R¹⁶, PR¹⁶R¹⁷, POR¹⁶R¹⁷, PO₂R¹⁶R¹⁷, OP(O)(OR¹⁷)₂,OCH₂OP(O)(OR¹⁷)₂, OC(O)OP(O)(OR¹⁷)₂, PO(OR¹⁶)(OR¹⁷),OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂, OC(O)R¹⁷ or OC(O)NHR¹⁷; wherein R¹⁴ and R¹⁵ areindependently H; linear or branched C₁-C₈ alkyl; C₂-C₈ alkenyl, alkynyl,heteroalkyl, heterocyclic, or carbocyclic; C₃-C₈ aryl, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl,or alkylcarbonyl; wherein when R¹⁴ is bivalent, R¹⁴ is further connectedto an additional functional group of one to four amino acid units, or(CH₂CH₂O)_(r), r is an integer ranging from 0 to 12, or C₄-C₁₂glycoside, or C₁-C₈ carboxylic acid; wherein R¹⁶ is H, OH, R¹⁴ or one tofour amino acid units; wherein R¹⁷ is H, linear or branched C₁-C₈ alkyl;C₂-C₈ alkenyl, alkynyl, heteroalkyl, or heterocyclic; C₃-C₈ aryl,carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl,heteroalkylcycloalkyl, heteroaralkyl, or alkylcarbonyl, or C₄-C₁₂glycoside, or a pharmaceutical salt.
 2. The conjugate according to claim1 having Formula (II):

or a pharmaceutically acceptable salt, hydrate, or hydrated saltthereof; or a polymorphic crystalline structure thereof; or an opticalisomer, racemate, diastereomer or enantiomer thereof; wherein T, L, n,m, Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R¹⁰, R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ aredefined the same as in the Formula (I); wherein R⁷ is H, R¹⁴,—R¹⁴C(═O)X¹R¹⁵; or —R¹⁴X¹R¹⁵; X¹ is O, S, S—S, NH, or NR¹⁴; wherein R⁹is H, —OH, —OR¹⁴, —OC(═O)R¹⁴, —OC(═O)NHR¹⁴, —OC(═O)NR¹⁴R¹⁵, —OC(═O)R¹⁴SSR¹⁵, OP(═O)(OR¹⁴)₂, or OR¹⁴OP(═O)(OR¹⁵); wherein R¹¹ is H, R¹⁴,—R¹⁴C(═O)R¹⁶, —R¹⁴C(═O)X²R¹⁶, —R¹⁴X²R¹⁶, or —R¹⁴C(═O)X², wherein X² is—O—, —S—, —NH—, —NHS(O₂), —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—, —S(═O)—R¹⁴—, or—NHR¹⁴—; wherein R¹² is H, R¹⁴, —O—, —S—, —N—, ═N—, ═NNH—, —OH, —SH,—NH₂, ═NH, ═NNH₂, —NH(R¹⁴), —OR¹⁴, —C(O)O—, —C(O)OR¹⁶—, —COR¹⁶,—COOR¹⁴—, C(O)NH—, C(O)NH₂, C(O)NHR¹⁴, —SR¹⁴, —S(═O)R¹⁴, —P(═O)(OR¹⁶)₂,—OP(═O)(OR¹⁶)₂, —CH₂OP(═O)(OR¹⁶)₂, or —SO₂R¹⁶.
 3. The conjugateaccording to claim 1 having Formula (III):

or a pharmaceutically acceptable salt, hydrate, or hydrated saltthereof; or a polymorphic crystalline structure thereof; or an opticalisomer, racemate, diastereomer or enantiomer thereof; wherein T, L, m,Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹, R¹³ and n are definedthe same as in Formula (I); wherein R⁷ is independently —R¹⁴—, or—R¹⁴C(═O)X¹R¹⁵— or —R¹⁴X¹R¹⁵—, wherein R¹⁴ and R¹⁵ are independentlylinear or branched C₁˜C₈ alkyl, or heteroalkyl; C₂˜C₈ alkenyl, oralkynyl; C₃˜C₈ aryl, heterocyclic, carbocyclic, cycloalkyl,heterocycloalkyl, heteroaralkyl heteroalkylcycloalkyl, or alkylcarbonyl;X¹ is O, S, S—S, NH, or NR¹⁴.
 4. The conjugate according to claim 1having Formula (IV):

or a pharmaceutically acceptable salt, hydrate, or hydrated saltthereof; or a polymorphic crystalline structure thereof; or an opticalisomer, racemate, diastereomer or enantiomer thereof; wherein T, L, m,R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁰, R¹¹, R¹², R¹³ and n are defined thesame as in Formula (I); wherein R⁹ is independently H, —O—, —OR¹⁴—,—OC(═O)R¹⁴—, —OC(═O)NHR¹⁴—, —OC(═O)NR¹⁴R¹⁵—, —OC(═O)R¹⁴SSR¹⁵—, or—OP(═O)(OR¹⁴)O—, wherein R¹⁴ and R¹⁵ are independently H, C₁˜C₈ alkyl,or heteroalkyl; C₃˜C₈ aryl, heteroaryl, heterocyclic, carbocyclic,cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl,heteroaralkyl, or alkylcarbonyl, or a pharmaceutical salt.
 5. Theconjugate according to claim 1 having Formula (V):

or a pharmaceutically acceptable salt, hydrate, or hydrated saltthereof; or a polymorphic crystalline structure thereof; or an opticalisomer, racemate, diastereomer or enantiomer thereof; wherein T, L, m,Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³ and n are definedthe same as in Formula (I); wherein R¹¹ is —R¹⁴—, —R¹⁴C(═O)R¹⁷—,—R¹⁴C(═O)X²R¹⁷—, —R¹⁴X²R¹⁷—, or —R₁₄C(═O)X²—, wherein R¹⁷ isindependently H, OH, C₁˜C₈ alkyl; C₂˜C₈ alkenyl, alkynyl, orheteroalkyl; C₃˜C₈ aryl, arylene, heterocyclic, carbocyclic, orheterocycloalkyl; or an amino acid, or two amino acid units; X² is —O—,—S—, —NH—, —NHS(O₂)—, —NHS(O)—, —N(R¹⁴)—, —O—R¹⁴—, —S—R¹⁴—, —S(═O)—R¹⁴—,or —NHR¹⁴—; —R¹⁴ is H, C₁˜C₈ alkyl, or heteroalkyl; C₂˜C₈ alkenyl, oralkynyl; C₃˜C₈ aryl, heterocyclic, carbocyclic, cycloalkyl,alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl,or alkylcarbonyl.
 6. The conjugate according to claim 1 having Formula(VI):

or a pharmaceutically acceptable salt, hydrate, or hydrated saltthereof; or a polymorphic crystalline structure thereof; or an opticalisomer, racemate, diastereomer or enantiomer thereof; wherein T, L, m,Y, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹¹, R¹³ and n are defined thesame as in Formula (I); wherein R¹² is independently R¹⁴, —O—, —S—,—NH—, ═N—, ═NNH—, —N(R₁₄)—, —OR¹⁴—, C(O)O—, C(O)NH—, C(O)NR¹⁴—, —SR¹⁴—,—S(═O)R¹⁴—, —NHR¹⁴—, —CH₂OP(═O)(OR¹⁵)—, —P(═O)(OR¹⁸)—, —OP(═O)(OR¹⁵)O—,or —SO₂R¹⁴, R¹⁴ and R¹⁵ are independently C₁˜C₈ alkyl, or heteroalkyl;C₂˜C₈ alkenyl, or alkynyl; C₃˜C₈ aryl, heterocyclic, carbocyclic,cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl,heteroalkylcycloalkyl, or alkylcarbonyl.
 7. The conjugate according toclaim 1 having Formula (VII):

or a pharmaceutically acceptable salt, hydrate, or hydrated salt; or apolymorphic crystalline structure thereof, or an optical isomer,racemate, diastereomer or enantiomer thereof, wherein T, L, n, m, Y, R¹,R^(1′), R², R³, R⁴, R⁶, R⁸, R¹⁰, R¹¹, and R¹² are defined the same as inFormula (I); wherein R¹³ is C₁˜C₁₀ alkyl, heteroalkyl, alkyl acid, alkylamide, alkyl amine, or Ar; Ar refers to a aromatic or hetero aromaticgroup, composed of one or several rings, comprising four to ten carbonatoms; the hetero aromatic group is an aromatic group wherein one, ormore carbon atoms are replaced by O, N, Si, Se, P or S, the aryl or Aris an aromatic group, wherein one or several H atoms are replacedindependently by R¹⁸, F, Cl, Br, I, OR¹⁶, SR¹⁶; NR¹⁶R¹⁸, N═NR¹⁶, N═R¹⁶,NR¹⁶R¹⁸, NO₂, SOR¹⁶R¹⁸, SO₂R¹⁶, SO₃R¹⁶, OSO₃R¹⁶, PR¹⁶R¹⁸, POR¹⁶R¹⁸,PO₂R¹⁶R¹⁸, OPO₃R¹⁶R¹⁸, or PO₃R¹⁶R¹⁸ wherein R¹⁶ and R¹⁸ areindependently H, C₁˜C₈ alkyl; C₂˜C₈ alkenyl, alkynyl, or heteroalkyl;C₃˜C₈ aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl,heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, oralkylcarbonyl; or C₄˜C₁₂ glycoside; or a pharmaceutical salt.
 8. Theconjugate according to claim 1, wherein the cytotoxic compound insidethe bracket of Formula (I) has a structure represented by one of thefollowing Formulae II-01˜II-69, III-01˜II-68, IV-01˜IV-68, V-01˜V-68,VI-01˜VI-12, and VII-01˜VII-77:

or a pharmaceutically acceptable salt, hydrate, or hydrated saltthereof; or a polymorphic crystalline structure thereof; or an opticalisomer, racemate, diastereomer or enantiomer thereof; wherein R²⁰ is H;C₁-C₈ linear or branched alkyl, heteroalkyl, or —C(O)R¹⁷; C₂-C₈ linearor branched alkenyl, alkynyl, alkylcycloalkyl, or heterocycloalkyl;C₃-C₈ linear or branched aryl, Ar-alkyl, heterocyclic, carbocyclic,cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl;—C(O)OR¹⁷, —C(O)NR¹⁷R¹⁸; or C₁-C₈ carboxylate, ester, ether, or amide;or 1˜8 amino acids; or polyethyleneoxy unit of formula (OCH₂CH₂)_(p) or(OCH₂CH(CH₃))_(p), wherein p is an integer from 0 to about 1000; or R²⁰is absent and the oxygen forms a ketone, or a combination of two or morethereof, wherein R²¹ is H, C₁-C₈ linear or branched alkyl; X, X¹, X²,and X³ are independently O, S, NH, NHNH, NHR¹⁷, CH₂ or absent; P¹ is H,R¹⁷, P(O)(OH)₂, P(O)(X¹R¹⁷)₂, CH₂P(O)(OH)₂, S(O²)(X¹R¹⁷), C₆H₁₂O₅, or(CH₂CH₂O)_(p)R¹⁷, wherein p is selected from 0-100, and R¹⁷ is definedabove; in addition X¹P¹ can be together; wherein Z² and Z³ areindependently H, OH, NH₂, OR¹⁷, NHR¹⁷, COOH, COOR¹⁷, C(O)R¹⁷, C(O)NHR¹⁷,C(O)NHNHR¹⁷, C(O)NH₂, R¹⁸, OCH₂OP(O)(OR¹⁸)₂, OC(O)OP(O)(OR¹⁸)₂,OPO(OR¹⁸)₂, NHPO(OR¹⁸)₂, OP(O)(OR¹⁸)OP(O)(OR¹⁸)₂, OC(O)R¹⁸, OC(O)NHR¹⁸,OSO₂(OR¹⁸), O—(C₄-C₁₂-glycoside), C₁-C₈ linear or branched alkyl orheteroalkyl; C₂-C₈ linear or branched alkenyl, alkynyl, alkylcycloalkyl,or heterocycloalkyl; C₃-C₈ linear or branched aryl, Ar-alkyl,heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl,alkylcarbonyl, or heteroaryl; —C(O)OR¹⁷, or —C(O)NR¹⁷R¹⁸; R¹⁷ and R¹⁸are independently H, C₁-C₈ linear or branched alkyl or heteroalkyl;C₂-C₈ linear or branched alkenyl, alkynyl, alkylcycloalkyl, orheterocycloalkyl; C₃-C₈ linear or branched aryl, Ar-alkyl, heterocyclic,carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, orheteroaryl; —C(O)OR¹⁷, or —C(O)NR¹⁷R¹⁸; R¹⁹ is H, OH, NH₂, OSO₂(OR¹⁸),XCH₂OP(O)(OR¹⁸)₂, XPO(OR¹⁸)₂, XC(O)OP(O)(OR¹⁸)₂, XC(O)R¹⁸, XC(O)NHR¹⁸,C₁˜C₈ alkyl or carboxylate; C₂˜C₈ alkenyl, alkynyl, alkylcycloalkyl, orheterocycloalkyl; C₃˜C₈ aryl or alkylcarbonyl; or a pharmaceutical salt;X is O, S, NH, NHNH, NHR⁷, or CH₂; R⁷ is defined the same above; wherein“

” is the site that linked to a linker L.
 9. The conjugate according toclaim 1 having one of the following structures:

wherein mAb is an antibody; n is 1˜20; and p is 0˜100.
 10. A method forpreparing the conjugate according to claim 9, comprising reacting theantibody with a compound having one of the following structures:


11. The conjugate according to claim 1, wherein the cell binding agentis capable of targeting against a tumor cell, a virus infected cell, amicroorganism infected cell, a parasite infected cell, an autoimmunedisease cell, an activated tumor cells, a myeloid cell, aw a T-cell, a Bcell, or a melanocyte, or a cell expressing any one of the followingantigens or receptors: CD2, CD2R, CD3, CD3gd, CD3e, CD4, CD5, CD6, CD7,CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD12w, CD13,CD14, CD15, CD15s, CD15u, CD16, CD16a, CD16b, CD17, CDw17, CD18, CD19,CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31,CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a,CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO,CD46, CD47, CD47R, CD48, CD49a, CD49b, CD49c, CD49e, CD49f, CD50, CD51,CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD60a, CD60b,CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a,CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72,CD73, CD74, CD74, CD75, CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b,CD80, CD81, CD82, CD83, CD84, CDw84, CD85, CD86, CD87, CD88, CD89, CD90,CD91, CD92, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R,CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b,CD108, CD109, CD110, CD111, CD112, CD113, CDw113, CD114, CD115, CD116,CD117, CD118, CD119, CDw119, CD120a, CD120b, CD121a, CD121b, CDw121b,CD122, CD123, CDw123, CD124, CD125, CDw125, CD126, CD127, CD128, CDw128,CD129, CD130, CD131, CDw131, CD132, CD133, CD134, CD135, CD136, CDw136,CD137, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144,CD145, CDw145, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153,CD154, CD155, CD156a, CD156b, CDw156c, CD157, CD158a, CD158b, CD159a,CD159b, CD159c, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166,CD167, CD167a, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g,CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179, CD180, CD181,CD182, CD183, CD184, CD185, CD186, CDw186, CD187, CD188, CD189, CD190,Cd191, CD192, CD193, CD194, CD195, CD196, CD197, CD198, CDw198, CD199,CDw199, CD200, CD200a, CD200b, CD201, CD202, CD202b, CD203, CD203c,CD204, CD205, CD206, CD207, CD208, CD209, CD210, CDw210, CD212, CD213a1,CD213a2, CDw217, CDw218a, CDw218b, CD220, CD221, CD222, CD223, CD224,CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234,CD235a, CD235ab, CD235b, CD236, CD236R, CD238, CD239, CD240, CD240CE,CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249,CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD265,CD266, CD267, CD268, CD269, CD271, CD273, CD274, CD275, CD276 (B7-H3),CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD289, CD292,CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c,CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD309, CD312, CD314,CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325,CD326, CDw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336,CD337, CDw338, CD339, CD340, CD341, CD342, CD343, CD344, CD345, CD346,CD347, CD348, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD356,CD357, CD358, CD359, CD360, CD361, CD362, CD363, CD364, CD365, CD366,CD367, CD368, CD369, CD370, CD371, CD372, CD373, CD374, CD375, CD376,CD377, CD378, CD379, CD381, CD382, CD383, CD384, CD385, CD386, CD387,CD388, CD389, CRIPTO, CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1,4-1BB, APO2, ASLG659, BMPR1B, 4-1BB, 5AC, 5T4 (Trophoblasticglycoprotein, TPBG, 5T4, Wnt-Activated Inhibitory Factor 1 or WAIF1),Adenocarcinoma antigen, AGS-5, AGS-22M6, Activin receptor-like kinase 1,AFP, AKAP-4, Alpha integrin, Alpha v beta6, Amino-peptidase N, Amyloidbeta, Angiopoietin 2, Angiopoietin 3, Annexin A1, AOC3 (VAP-1), B7-H3,Bacillus anthracis anthrax, BAFF, BCMA, B-lymphoma cell, Bombesin, C5,C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX,carbonic anhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31,Carbonic anhydrase IX, Cardiac myosin, CCL11(C—C motif chemokine 11),CCR4 (C—C chemokine receptor type 4), CCR5, CD3E (epsilon), CEA(Carcinoembryonic antigen), CEACAM3, CEACAM5 (carcino-embryonicantigen), CFD (Factor D), Cholecystokinin 2 (CCK2R), CLDN18(Claudin-18), CLDN18.1 (Claudin-18.1), CLDN18.2 (Claudin-18.2), Clumpingfactor A, cMet, CRIPTO, FCSF1R (Colony stimulating factor 1 receptor),CSF2 (colony stimulating factor 2, Granulocyte-macrophagecolony-stimulating factor (GM-CSF)), CSP4, CTLA4 (cytotoxicT-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4,C-X-C chemokine receptor type 4, cyclic ADP ribose hydrolase,Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran, DLL3(delta-like-ligand 3), DLL4 (delta-like-ligand 4), DPP4(Dipeptidyl-peptidase 4), DR5 (Death receptor 5), ED-B, EGFL7 (EGF-likedomain-containing protein 7), EGFR, EGFRII, EGFRvIII, Endoglin,Endothelin B receptor, Endotoxin, EpCAM (epithelial cell adhesionmolecule), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2),ERBB3, ERG, Escherichia coli, ET-V6-AML-FAP (Fibroblast activationprotein alpha), fibroblast surface antigen, FCGR1, alpha-Fetoprotein,Fibrin II, beta chain, Fibronectin extra domain-B, FOLR (folatereceptor), Folate receptor alpha, Folate hydrolase, Fos-related antigen,1F protein of respiratory syncytial virus, Frizzled receptor, FucosylGM1, GD2 ganglioside, G-28 GloboH, Glypican 3, N-glycolylneuraminicacid, GM3, GMCSF receptor α-chain, Growth differentiation factor,GP-14-GPNMB (Trans-membrane glycoprotein NMB), GUCY2C (Guanylate cyclase2C, guanylyl cyclase C(GC-C), intestinal Guanylate cyclase, Guanylatecyclase-C receptor, Heat-stable enterotoxin receptor), Heat shockproteins, Hemagglutinin, Hepatitis B surface antigen, Hepatitis B virus,HER1 (human epidermal growth factor receptor 1), HER2, HER2/neu, HER3(ERBB-3), IgG4, HGF/SF (Hepatocyte growth factor/scatter factor), HHGFR,HIV-1, HLA-HLA-DR10, HLA-DRB, HMWMAA, Human chorionic gonadotropin,HNGF, Human scatter factor receptor kinase, Hsp90, ICAM-1 (IntercellularAdhesion Molecule 1), Idiotype, IGF1R (insulin-like growth factor 1receptor), IFN-γ, Influenza hemagglutinin, IgE, IgE Fc region,interleukins of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-17A, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-27, or IL-28), IL31RA, ILGF2 (Insulin-likegrowth factor 2), Integrins (α4, α_(IIb)β₃, αvβ3, α₄β₇, α5β1, α60β,α7β7, αllβ3, α5β5, αvβ5), Interferon gamma-induced protein, ITGA2,ITGB2, KIR2D, Kappa Ig, Legumain, Lewis-Y antigen, LFA-1 (Lymphocytefunction-associated antigen 1, CD11a), LHRH, LINGO-1, Lipoteichoic acid,LIV1A, LTA, MAGE-1, MAGE-2, MAGE-3, MAGE 4, MART1, MCP-1, MIF(Macrophage migration inhibitory factor, or glycosylation-inhibitingfactor (GIF)), MS4A1 (membrane-spanning 4-domains subfamily A member 1),MSLN (mesothelin), Mucin 1, cell surface associated (MUC1) orpolymorphic epithelial mucin (PEM), MUC16 (CA125), MCP1 (monocytechemotactic protein 1), MPG, MS4A1 (membrane-spanning 4-domainssubfamily A), Myelin-associated glycoprotein, Myostatin, NARP-1, NCA-90,Nectin-4 (ASG-22ME), NGF, Neural apoptosis-regulated proteinase 1,NOGO-A, Notch receptor, Nucleolin, Neu oncogene product, NY-BR-1, OX-40,OxLDL (Oxidized low-density lipoprotein), P21, P97, PAP, PCSK9, PDCD1(PD-1, Programmed cell death protein 1), PDGF-Rα (Alpha-typeplatelet-derived growth factor receptor), PDGFR-β, PDL-1, PLAC1,PLAP-like testicular alkaline phosphatase, Platelet-derived growthfactor receptor beta, Phosphate-sodium co-transporter, PMEL 17,Polysialic acid, Prostatic carcinoma, PS (Phosphatidylserine), Prostaticcarcinoma cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies virusglycoprotein, RHD (Rh polypeptide 1 (RhPI)), Rhesus factor, RANKL,ROBO4, Respiratory syncytial virus, RON, ROR1, Sclerostin, SLAMF7 (SLAMfamily member 7), Selectin P, SDC1 (Syndecan 1), sLe(a), Somatomedin C,SIP (Sphingosine-1-phosphate), Somatostatin, Sperm protein 17, STEAP1(six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn,TAG-72 (tumor associated glycoprotein 72), T-cell receptor, T celltransmembrane protein, TEM1 (Tumor endothelial marker 1), TENB2,Tenascin C (TN-C), TGF-α, TGF-β (Transforming growth factor beta),TGF-β1, TGF-β2 (Transforming growth factor-beta 2), Tie (CD202b), Tie2,TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosisfactor receptor superfamily member 10B), TNFRSF-13B (tumor necrosisfactor receptor superfamily member 13B), TPBG (trophoblastglycoprotein), TRAIL-R₁ (Tumor necrosis apoptosis Inducing ligandReceptor 1), TRAILR2 (Death receptor 5 (DR5)), tumor-associated calciumsignal transducer 2, tumor specific glycosylation of MUC1, TWEAKreceptor, TYRP1 (glycoprotein 75), TRP-1 (Trop1), Trop2, VCAM-1, VEGF,VEGF-A, VEGF-2, VEGFR-1, VEGFR2, or vimentin, or a cell expressing aninsulin growth factor receptor, or an epidermal growth factor receptor.12. The conjugate according to claim 11, wherein the tumor cell isselected from the group consisting of lymphoma cells, myeloma cells,renal cells, breast cancer cells, prostate cancer cells, ovarian cancercells, colorectal cancer cells, gastric cancer cells, squamous cancercells, small-cell lung cancer cells, none small-cell lung cancer cells,testicular cancer cells, malignant cells, and cells that grow and divideat an unregulated, quickened pace to cause cancers.
 13. A pharmaceuticalcomposition comprising a therapeutically effective amount of one or moreof the conjugate of claim 1, and a pharmaceutically acceptable salt,carrier, diluent, or excipient therefor.
 14. The pharmaceuticalcomposition of claim 13, having in vitro, in vivo or ex vivo cellkilling activity.
 15. A method for treating a cancer, an autoimmunedisease, or an infectious disease, comprising administering concurrentlythe pharmaceutical composition a according to claim 13, with a agentselected from chemotherapeutic agent, a radiation therapy, animmunotherapy agent, an autoimmune disorder agent, or an anti-infectiousagents-.
 16. The method according to claim 15, wherein the agent is oneor more selected from the following agents: (1) a chemotherapeutic agentconsisting of: a) an alkylating agent: selected from the groupconsisting of nitrogen mustards: chlorambucil, chlornaphazine,cyclophosphamide, dacarbazine, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine,mitobronitol, melphalan, mitolactol, pipobroman, novembichin,phenesterine, prednimustine, thiotepa, trofosfamide, uracil mustard;CC-1065 and adozelesin, carzelesin, bizelesin and their syntheticanalogues; duocarmycin and its synthetic analogues, KW-2189, CBI-TMI,CBI dimers; benzodiazepine dimers and pyrrolobenzodiazepine (PBD)dimers, tomaymycin dimers, indolinobenzodiazepine dimers,imidazobenzothiadiazepine dimers, and oxazolidinobenzodiazepine dimers;Nitrosoureas: comprising carmustine, lomustine, chlorozotocin,fotemustine, nimustine, ranimustine; Alkylsulphonates: comprisingbusulfan, treosulfan, improsulfan and piposulfan); Triazenes anddacarbazine; Platinum containing compounds: comprising carboplatin,cisplatin, and oxaliplatin; aziridines, benzodopa, carboquone,meturedopa, or uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphoramide and trimethylolomelamine; b) a plantalkaloid: selected from the group consisting of Vinca alkaloids:comprising vincristine, vinblastine, vindesine, vinorelbine, andnavelbin; Taxoids of paclitaxel, docetaxol and their analogs,Maytansinoids of DM1, DM2, DM3, DM4, DM5, DM6, DM7, maytansine,ansamitocins and their analogs, cryptophycin 1, cryptophycin 8;epothilones, eleutherobin, discodermolide, bryostatins, dolostatins,auristatins, tubulysins, cephalostatins; pancratistatin; a sarcodictyin;and spongistatin; c) a DNA topoisomerase inhibitor: selected from thegroup consisting of Epipodophyllins, 9-aminocamptothecin, camptothecin,crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan,mitoxantrone, novantrone, retinoic acids, retinols, teniposide,topotecan, 9-nitrocamptothecin, RFS 2000; and mitomycins and theiranalogs; d) an antimetabolite: selected from the group consisting ofmethotrexate, trimetrexate, denopterin, pteropterin, aminopterin andfolic acid analogues; mycophenolic acid, tiazofurin, ribavirin, EICAR;hydroxyurea, deferoxamine; Pyrimidine analogs of Uracil, analogs ofancitabine, azacytidine, 6-azauridine, capecitabine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-Fluorouracil,floxuridine, ratitrexed; Cytosine analogs of cytarabine, cytosinearabinoside, and fludarabine; Purine analogs of azathioprine,fludarabine, mercaptopurine, thiamiprine, and thioguanine; folic acidreplenisher, and folinic acid; e) a hormonal therapy selected from thegroup consisting of Anti-estrogen of megestrol, raloxifene, andtamoxifen; goserelin, leuprolide acetate; bicalutamide, flutamide,calusterone, dromostanolone propionate, epitiostanol, goserelin,leuprolide, mepitiostane, nilutamide, testolactone, trilostane VitaminD3 analogs of CB 1093, EB 1089, KH 1060, cholecalciferol, andergocalciferol; verteporfin, phthalocyanine, photosensitizer Pc4,demethoxyhypocrellin A; Cytokines of Interferon-alpha, Interferon-gamma,tumor necrosis factor (TNFs), and human proteins containing a TNFdomain; f) a kinase inhibitor, selected from the group consisting ofBIBW 2992, imatinib, gefitinib, pegaptanib, sorafenib, dasatinib,sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib,vandetanib, E7080, mubritinib, ponatinib (AP24534), bafetinib(INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib,ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab,cetuximab, Trastuzumab, Ranibizumab, Panitumumab, and ispinesib; g) apoly (ADP-ribose) polymerase (PARP) inhibitors selected from the groupconsisting of olaparib, niraparib, iniparib, talazoparib, veliparib, CEP9722, E7016, BGB-290, and 3-aminobenzamide; h) an antibiotic, selectedfrom the group consisting of an enediyne antibiotic (selected fromtocalicheamicin, calicheamicin γ1, δ1, α1 and β1; dynemicin A,deoxydynemicin; esperamicin, kedarcidin, C-1027, maduropeptin,neocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromomophores), aclacinomycins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin; chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin,epirubicin, eribulin, esorubicin, idarubicin, marcellomycin, nitomycins,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, and zorubicin; i) a polyketide,bullatacin and bullatacinone; gemcitabine, epoxomicins and carfilzomib,bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat,zybrestat, PLX4032, STA-9090, a Stimuvax, allovectin-7, Xegeva,Sipuleucel-T, ipilimumab, Isoprenylation inhibitors and Lovastatin,Dopaminergic neurotoxins and 1-methyl-4-phenylpyridinium ion,staurosporine, Actinomycin D, dactinomycin, amanitins, bleomycin A2,bleomycin B2, peplomycin, daunorubicin, doxorubicin (adriamycin),idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil,thapsigargin, Vorinostat, Romidepsin, Panobinostat, Valproic acid,Mocetinostat (MGCD0103), Belinostat, PCI-24781, Entinostat, SB939,Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, Trichostatin A);Thapsigargin, Celecoxib, glitazones, epigallocatechin gallate,Disulfiram, Salinosporamide A; Anti-adrenals, selected from the groupconsisting of aminoglutethimide, mitotane, trilostane; aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside,bestrabucil; bisantrene; edatrexate; defofamine; demecolcine;diaziquone; eflornithine (DFMO), elfomithine; elliptinium acetate,etoglucid; gallium nitrate; gacytosine, hydroxyurea; ibandronate,lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine;pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; T-2 toxin, verrucarin A, roridin A,anguidine; urethane, siRNA, antisense drugs; (2) an anti-autoimmunedisease agent: cyclosporine, cyclosporine A, aminocaproic acid,azathioprine, bromocriptine, chlorambucil, chloroquine,cyclophosphamide, amcinonide, betamethasone, budesonide, hydrocortisone,flunisolide, fluticasone propionate, fluocortolone danazol,dexamethasone, Triamcinolone acetonide, beclometasone dipropionate,DHEA, enanercept, hydroxychloroquine, infliximab, meloxicam,methotrexate, mofetil, mycophenylate, prednisone, sirolimus, tacrolimus(3) an anti-infectious disease agents comprising: a) aminoglycosides:amikacin, astromicin, netilmicin, sisomicin, isepamicin, hygromycin B,amikacin, arbekacin, bekanamycin, dibekacin, tobramycin, framycetin,paromomycin, ribostamycin, netilmicin, spectinomycin, streptomycin,tobramycin, verdamicin; b) amphenicols: azidamfenicol, chloramphenicol,florfenicol, thiamphenicol; c) ansamycins: geldanamycin, herbimycin; d)carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin,meropenem, panipenem; e) cephems: carbacephem, cefacetrile, cefaclor,cefradine, cefadroxil, cefalonium, cefaloridine, cefalotin orcefalothin, cefalexin, cefaloglycin, cefamandole, cefapirin,cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone,cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil,cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren,cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone,ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimizole,cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin,ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime,ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cefoxitin, cefotetan,cefmetazole, flomoxef, latamoxef; f) glycopeptides: bleomycin,oritavancin, telavancin, teicoplanin, ramoplanin; g) tigecycline; h)β-lactamase inhibitors: sulbactam, tazobactam, clavulanic acid; i)Lincosamides: clindamycin, lincomycin; j) lipopeptides: daptomycin,A54145, calcium-dependent antibiotics (CDA); k) macrolides:azithromycin, cethromycin, clarithromycin, dirithromycin, erythromycin,flurithromycin, josamycin, telithromycin, cethromycin, midecamycin,miocamycin, oleandomycin, rifampicin, rifampin, rifabutin, rifapentine),rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus(FK506), troleandomycin, telithromycin; l) monobactams: aztreonam,tigemonam; m) oxazolidinones: linezolid; n) penicillins: amoxicillin,ampicillin, pivampicillin, hetacillin, bacampicillin, metampicillin,talampicillin, azidocillin, azlocillin, benzylpenicillin, benzathinebenzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin,procaine benzylpenicillin, carbenicillin, cloxacillin, dicloxacillin,epicillin, flucloxacillin, mecillinam, mezlocillin, meticillin,nafcillin, oxacillin, penamecillin, penicillin, pheneticillin,phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin,temocillin, ticarcillin; o) polypeptides: bacitracin, colistin,polymyxin B; p) quinolones: alatrofloxacin, balofloxacin, ciprofloxacin,clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxin,garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, kanotrovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin,nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin,trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin,tosufloxacin, trovafloxacin; q) streptogramins: pristinamycin,quinupristin/dalfopristin; r) sulfonamides: mafenide, prontosil,sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine,sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole(co-trimoxazole); s) steroid antibacterials: selected from fusidic acid;t) tetracyclines: doxycycline, chlortetracycline, clomocycline,demeclocycline, lymecycline, meclocycline, metacycline, minocycline,oxytetracycline, penimepicycline, rolitetracycline, tetracycline,tigecycline; u) antibiotics: selected from the group consisting ofannonacin, arsphenamine, Bacitracin, cycloserine, dictyostatin,discodermolide, eleutherobin, epothilone, ethambutol, etoposide,faropenem, fusidic acid, furazolidone, isoniazid, laulimalide,metronidazole, mupirocin, fosfomycin, nitrofurantoin, paclitaxel,platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin(rifampin), tazobactam tinidazole, uvaricin; (4) Anti-viral drugscomprising: a) entry/fusion inhibitors: aplaviroc, maraviroc,vicriviroc, gp41, PRO 140, CD4; b) integrase inhibitors: raltegravir,elvitegravir, globoidnan A; c) maturation inhibitors: bevirimat,vivecon; d) neuraminidase inhibitors: oseltamivir, zanamivir, peramivir;e) nucleosides & nucleotides: abacavir, aciclovir, adefovir, amdoxovir,apricitabine, brivudine, cidofovir, clevudine, dexelvucitabine,didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir,famciclovir, fluorouracil (5-FU), 3′-fluoro-substituted2′,3′-dideoxynucleoside analogues of 3′-fluoro-2′,3′-dideoxythymidine(FLT) and 3′-fluoro-2′,3′-dideoxyguanosine (FLG), fomivirsen,ganciclovir, idoxuridine, lamivudine (3TC), 1-nucleosides ofβ-1-thymidine and β-1˜2′-deoxycytidine, penciclovir, racivir, ribavirin,stampidine, stavudine (d4T), taribavirin (viramidine), telbivudine,tenofovir, trifluridine valaciclovir, valganciclovir, zalcitabine (ddC),zidovudine (AZT); f) non-nucleosides: amantadine, ateviridine,capravirine, diarylpyrimidines of etravirine and rilpivirine,delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformicacid), imiquimod, interferon alfa, loviride, lodenosine, methisazone,nevirapine, NOV-205, peginterferon alfa, podophyllotoxin, rifampicin,rimantadine, resiquimod (R-848), tromantadine; g) protease inhibitors:amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir,lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir(VX-950), tipranavir; h) Anti-virus drugs: abzyme, arbidol, calanolidea, ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate(EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea,KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin,seliciclib; (5) a radioisotope for radiotherapy selected from the groupconsisting of (radionuclides)³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga,⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, and²¹³Bi; (6) a cell-binding molecule-drug conjugate having a cytotoxicagent of a tubulysin analog, maytansinoid analog, taxanoid (taxane)analog, CC-1065 analog, daunorubicin and doxorubicin compound, amatoxinanalog, dimer of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin,indolinobenzodiazepines, imidazobenzothiadiazepines, oroxazolidinobenzodiazepines, calicheamicins and the enediyne antibioticcompound, actinomycin, azaserine, bleomycins, epirubicin, tamoxifen,idarubicin, dolastatins, auristatins of monomethyl auristatin E, MMAE,MMAF, auristatin PYE, auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB),and EFP (AEFP), duocarmycins, geldanamycins, methotrexates, thiotepa,vindesines, vincristines, hemiasterlins, nazumamides, microginins,radiosumins, topoisomerase I inhibitors, alterobactins,microsclerodermins, theonellamides, esperamicins, PNU-159682, and theiranalogues and derivatives above thereof; (7) a pharmaceuticallyacceptable salt, acid or derivative of any of the above drugs (1) to(6).
 17. The conjugate according to claim 1, wherein T is an antibody; asingle chain antibody; an antibody fragment that binds to the targetcell; a monoclonal antibody; a single chain monoclonal antibody; or amonoclonal antibody fragment that binds the target cell; a chimericantibody; a chimeric antibody fragment that binds to the target cell; adomain antibody; or a domain antibody fragment that binds to the targetcell.